Characterization of an ATP-dependent DNA strand transferase from human cells.

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.     

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.     

Study of the photocycle and charge motions of the bacteriorhodopsin mutant D96N.

Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.     

The inner interhelix loop 4-5 of the melibiose permease from Escherichia coli takes part in conformational changes after sugar binding

Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.     

Protection against glycation and similar post-translational modifications of proteins

Glycation and other non-enzymic post-translational modifications of proteins have been implicated in the complications of diabetes and other conditions. In recent years there has been extensive progress in the search for ways to prevent the modifications and prevent the consequences of the modifications. These areas are covered in this review together with newer ideas on possibilities of reversing the chemical modifications.     

Inhibition of endotoxin-induced macrophage chemokine production by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in vitro and in vivo.

Inflammatory chemokines recruit various populations of immune cells that initiate and maintain the inflammatory response against foreign Ags. Although such a response is necessary for the elimination of the Ag, the inflammation has to be eventually resolved in a healthy organism. Neuropeptides such as vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), released after antigenic stimulation, contribute to the termination of an inflammatory response primarily by inhibiting the production of proinflammatory cytokines. Here we investigated the effects of VIP and PACAP on chemokine production. We report that VIP and PACAP inhibit the expression of the macrophage-derived CXC chemokines macrophage inflammatory protein-2 and KC (IL-8), and of the CC chemokines MIP-1alpha, MIP-1beta, monocyte chemoattractant protein 1, and RANTES in vivo and in vitro. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NF-kappaB binding and transactivating activity. The VIP/PACAP inhibition of both chemokine production and of NF-kappaB binding and transactivating activity is mediated through the specific VIP receptor VPAC1, and involves both cAMP-dependent and -independent intracellular pathways. In an in vivo model of acute peritonitis, the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the recruitment of polymorphonuclear cells, macrophages, and lymphocytes into the peritoneal cavity. These findings support the proposed role of VIP and PACAP as key endogenous anti-inflammatory agents and describe a novel mechanism, i.e., the inhibition of the production of macrophage-derived chemokines.     

Repair of single-stranded DNA nicks, gaps, and loops in mammalian cells.

We studied the ability of mammalian cells to repair single-stranded nicks, gaps, and loops in DNA duplexes. Heteroduplexes prepared from derivatives of the shuttle vector pSV2neo were introduced into monkey COS cells. After replication, the plasmids were recovered and used to transform Escherichia coli. Plasmid DNA from the recovered colonies was tested for repair at each of six different sites. We observed that mammalian cells are capable of repairing single-stranded gaps and free single-stranded ends most efficiently. Regions containing twin loops were recognized, and one of the loops was excised. Portions of the molecules containing small single loops were also repaired. Markers which were 58 nucleotides apart were corepaired with nearly 100% efficiency, while markers which were 1,000 nucleotides or more apart were never corepaired. The mechanisms involved in heteroduplex repair in mammalian cells seem to be similar to those involved in repairing DNA lesions caused by physical and chemical agents.