Abstract: The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties. To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1 transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs, which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone properties are mostly supported by its basic clusters.
Abstract: Probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research and membrane biophysics. Previous studies showed differences in accumulation and intracellular activity between two fluoroquinolones, ciprofloxacin and moxifloxacin, that may actually result from their differential susceptibility to efflux by the ciprofloxacin transporter. In view of the critical role of lipids for the drug cellular uptake and differences observed for the two closely related fluoroquinolones, we investigated the interactions of these two antibiotics with lipids, using an array of complementary techniques. Moxifloxacin induced, to a greater extent than ciprofloxacin, an erosion of the DPPC domains in the DOPC fluid phase (atomic force microscopy) and a shift of the surface pressure-area isotherms of DOPC/DPPC/fluoroquinolone monolayer toward lower area per molecule (Langmuir studies). These effects are related to a lower propensity of moxifloxacin to be released from lipid to aqueous phase (determined by phase transfer studies and conformational analysis) and a marked decrease of all-trans conformation of acyl-lipid chains of DPPC (determined by ATR-FTIR) without increase of lipid disorder and change in the tilt between the normal and the germanium surface (also determined by ATR-FTIR). All together, differences of ciprofloxacin as compared to moxifloxacin in their interactions with lipids could explain differences in their cellular accumulation and susceptibility to efflux transporters.
Abstract: The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.
Abstract: The HCV core protein plays a critical role in several steps of the viral cycle, notably in the encapsidation. Almost of its activities are relied on its interactions with intracellular protein and viral RNA. Recent studies on interactions of core protein and viral genome of HCV shown that basic domains presents in the N-terminal of HCV core protein are mainly involved in RNA binding1.2. Furthermore, the core protein has been shown to present analogy with the HIV nucleocapsid protein (NCp7). Similar to NC, HCV core protein facilities the annealing of cTAR/TAR of HIV sequences. However, specific details on chaperone properties of core HCV protein and the mechanism by which this protein facilities the annealing of complementary sequences have not been elucidated. In this study, we investigate the chaperone properties of basic domains {E (2BD), F (3BD)} and the tryptophan rich domain { A (74-121) } of HCV core protein, on native and mutated cTAR derivatives, as a models, by fluorescence spectroscopy, in three steps: Firstly we investigate its interaction with nucleic acids. Secondly, the destabilizing effects on the nucleic acids, and finally, we investigated the annealing kinetics of complementary sequences.
The binding parameters of E (2BD) HCV peptide with cTAR derivatives were determined by steady state anisotropy fluorescence. The stoichiometry of the binding is found to be about 3 and 2 peptides per cTAR and cTAR (14-39) respectively, suggesting a complete coating of the oligonucliotide by the peptide. The binding constants of E (2BD) HCV peptide with cTAR derivatives are in the order of 106M-1, indicating a lack of sequence specificity. In contrast to HIV nucleocapsid protein (NC), the core protein is unable to destabilize the secondary structure of cTAR, probably due to the absence of zinc finger structure in these peptides. However, the core protein (peptide E, F and A) strongly stimulate the annealing of cTAR/TAR several order of magnitude than their annealing without protein. The annealing rate constant depends on: i) The presence of the basic domain which ensuring the binding of core with oligonucliotides ii) and the stability of both TAR and cTAR sequence.
The annealing of TAR with its complementary sequence involves two second-order kinetic components that are activated by core HCV peptides. Moreover, it seems that the two kinetics components are correlate with core’s ability to bind on the loop and the lower half of TAR stem (data not shown). Finally, our findings provide valuable information for further investigations on chaperon properties of HCV core protein.
Abstract: The nucleocapsid protein NCp7 plays a critical role in several steps of the viral cycle, notably in the encapsidation. Almost all of its activities are relied on interaction with nucleic acids. Indeed NCp7 is thought to specifically interact with ψ encapsidation sequence of the genomic RNA, enabling its selection from the pool of cellular RNAs and its packaging (1). Previous studies of interaction between NCp7 and short sequences of the HIV-1 ψ packaging signal have shown that NC binds with high affinity to the loop of stem loops (3SL) (2,3). Furthermore, binding of NCp7 to short single strand hexanucleotides have been investigated in detail in our laboratory, indicating that G residue regardless to its location in the oligonucleotide appears essential for high affinity binding, via its stacking with tryptophane (Trp 37) of the NC protein (4,5). To further identify the key nucleotide in the formation of NCp7- nucleic acids complexes, we follow its interaction with oligonucleotides containing twelve bases and characterised by the presence of TG motif in different locations by steady state and time resolved anisotropy. Our results show that all sequences tested bind one protein molecule, even though the presence of two potential sites of binding, with a similar affinity within the range of K app= 3.5-4 106 M-1 probably due to a steric hindrance or negative cooperativity. If TG motif is on 5’ in d(TGAAACAACGAC) then the binding affinity decreases, indicating a critical role of TG motif in the NCp7/ oligonucleotide interaction. Interesting, the presence of CG motif located in close proximity to potential site of binding, stabilizes weakly the interaction with NCp7 by about (ΔΔG = -0.54Kcal/mol). This implies that (TGACCG) sequence appears to be the optimal sequence for binding of NCp7. Finally, the investigation of binding mechanism between NCp7 and HIV-1 ψ sequence in encapsidation process appears to be a key step to develop a novel strategy to inhibit this interaction.
