16 research outputs found
Multi-step purification strategy for RANTES wild-type and mutated analogues expressed in a baculovirus system
RANTES (regulated on activation, normal T cell expressed and secreted), a C-C chemokine, is one of the major HIV-suppressive factors produced by CD8+ T cells. Wild-type RANTES and genetically modified analogues were expressed in a baculovirus system and purified from cell culture supernatants employing a multi-step strategy based on affinity and RP-HPLC. Quantification and purity control of the final proteins were carried out by capillary electrophoresis using the synthetic or the recombinant wild-type RANTES as a reference. The procedure here reported requires only three days to obtain 0.016-0.270 mg of the pure and characterised proteins, starting from 370-900 ml of culture media, and is suitable for the analysis of a large number of RANTES analogues
Structural determinants of CCR5 recognition and HIV-1 blockade in RANTES
Certain chemokines act as natural antagonists of human immunodeficiency virus (HIV) by blocking key viral coreceptors, such as CCR5 and CXCR4, on the surface of susceptible cells. Elucidating the structural determinants of the receptor-binding and HIV-inhibitory functions of these chemokines is essential for the rational design of derivative molecules of therapeutic value. Here, we identify the structural determinants of CCR5 recognition and antiviral activity of the CC chemokine RANTES, showing that critical residues form a solvent-exposed hydrophobic patch on the surface of the molecule. Moreover, we demonstrate that the biological function is critically dependent on dimerization, resulting in the exposure of a large (180 \uc52), continuous hydrophobic surface. Relevant to the development of novel therapeutic approaches, we designed a retroinverted RANTES peptide mimetic that maintained both HIV- and chemotaxis-antagonistic functions
Enhancement of the HIV-1 inhibitory activity of RANTES by modification of the N-terminal region: dissociation from CCR5 activation
Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infection, their inherent proinflammatory activity may limit a therapeutic use. To elucidate whether the antiviral and signaling functions of RANTES can be dissociated, several recombinant analogues mutated at the N terminus were generated and functionally compared with the wild-type (WT) molecule, as well as with three previously described mutants. Substitution of selected residues within the N-terminal region caused a marked loss of antiviral potency. By contrast, two unique analogues (C1.C5-RANTES and L-RANTES) exhibited an increased antiviral activity against different CXCR4-negative HIV-1 isolates grown in primary mononuclear cells or in macrophages. This enhanced HIV-blocking activity was associated with an increased binding affinity for CCR5. Both C1.C5-RANTES and L-RANTES showed a dramatically reduced ability to trigger intracellular calcium mobilization via CCR3 or CCR5, while potently antagonizing the action of the WT chemokine. By contrast, two previously described analogues (RANTES(3-68) and AOP-RANTES) maintained a WT ability to trigger CCR5-mediated signaling, while a third one (RANTES(9-68)) showed a dramatic loss of antiviral activity. These data demonstrate that the antiviral and signaling functions of RANTES can be uncoupled, opening new perspectives for the development of chemokine-based therapeutic approaches for HIV infection
Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin.
The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex
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Elucidating a Key Anti-HIV-1 and Cancer-Associated Axis: The Structure of CCL5 (Rantes) in Complex with CCR5
CCL5 (RANTES) is an inflammatory chemokine which binds to chemokine receptor CCR5 and induces signaling. The CCL5:CCR5 associated chemotactic signaling is of critical biological importance and is a potential HIV-1 therapeutic axis. Several studies provided growing evidence for the expression of CCL5 and CCR5 in non-hematological malignancies. Therefore, the delineation of the CCL5:CCR5 complex structure can pave the way for novel CCR5-targeted drugs. We employed a computational protocol which is primarily based on free energy calculations and molecular dynamics simulations, and report, what is to our knowledge, the first computationally derived CCL5:CCR5 complex structure which is in excellent agreement with experimental findings and clarifies the functional role of CCL5 and CCR5 residues which are associated with binding and signaling. A wealth of polar and non-polar interactions contributes to the tight CCL5:CCR5 binding. The structure of an HIV-1 gp120 V3 loop in complex with CCR5 has recently been derived through a similar computational protocol. A comparison between the CCL5 : CCR5 and the HIV-1 gp120 V3 loop : CCR5 complex structures depicts that both the chemokine and the virus primarily interact with the same CCR5 residues. The present work provides insights into the blocking mechanism of HIV-1 by CCL5