Interplay between glutathione,Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1

Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu–ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log β) of the Cu(I) complexes measured at pH 6 were 15–16 and 49–50 for CuAtx1 and Cu₂^I(GS⁻)₂(Atx1)₂, respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu₂^I(GS⁻)₂(Atx1)₂ is the major conformation of Atx1 in the cytosol.     

Reversal of the sarcoplasmic reticulum ATPase cycle by substituting various cations for magnesium. Phosphorylation and ATP synthesis when Ca2+ replaces Mg2+

Reversal of the cycle of sarcoplasmic reticulum ATPase starts from ATPase phosphorylation by Pi, in the presence of Mg2+, and leads to ATP synthesis. We show here that ATP can also be synthesized when Ca2+ replaces Mg2+. In the absence of a calcium gradient and in the presence of dimethyl sulfoxide, ATPase phosphorylation from Pi and Ca2+ led to the formation of an unstable phosphoenzyme. This instability was due to a competition between the phosphorylation reaction induced by Pi and Ca2+ and the transition induced by Ca2+ binding to the transport sites, which led to a conformation that could not be phosphorylated from Pi. Dimethyl sulfoxide and low temperature stabilized the calcium phosphoenzyme, which under appropriate conditions, subsequently reacted with ADP to synthesize ATP. Substitution of Co2+, Mn2+, Cd2+, or Ni2+ for Mg2+ induced ATPase phosphorylation from Pi, giving phosphoenzymes of various stabilities. However, substitution of Ba2+, Sr2+, or Cr3+ produced no detectable phosphoenzymes, under the same experimental conditions. Our results show that ATPase phosphorylation from Pi, like its phosphorylation from ATP, does not have a strict specificity for magnesium.     

Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA)

Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP–HspA binds nickel ions with an apparent dissociation constant (K_d) of 1.8 μM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99–100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.     

Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.     

Cytokine regulation of metalloproteinase gene expression

Matrix metalloproteinases belong to a family of zinc-dependent enzymes capable of degrading extracellular matrix and basement membrane components. Their expression is greatly modulated by cytokines and growth factors and involves the gene products of the Fos and Jun families of oncogenes. After extra(peri)cellular activation, their activity can be further controlled by specific tissue inhibitors of metalloproteinases. A correct balance between these regulatory mechanisms is necessary to ensure matrix remodeling in normal physiological processes such as embryonic development, but the overexpression of these enzymes may initiate or contribute to pathological situations such as cartilage degradation in rheumatoid arthritis or to tumor progression and metastasis. Delineation of the mechanisms of metalloproteinase and metalloproteinase inhibitors gene expression, understanding of their mode of interactions, and characterization of their patterns of expression in various tissues in normal and pathological states will lead to new therapeutic strategies to counteract the deleterious effects of matrix metalloproteinases in human disease.     

Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosinetriphosphatase as reflected by organic solvent induced perturbation

The mechanism of sarcoplasmic reticulum (SR) ATPase Mg2+-dependent phosphorylation from Pi was investigated in the presence of 15% v/v dimethyl sulfoxide at pH 6, 20 degrees C, and in the absence of potassium. Measurements of intrinsic fluorescence changes and of 32P-labeled phosphoprotein (*E-P) were in agreement, both at equilibrium and in transient situations. We found that the amount of phosphoenzyme present and its rate of formation depended solely on the concentration of the (Mg X Pi) complex. Up to 6 nmol of phosphate/mg of protein was covalently bound to the enzyme, implying almost complete phosphorylation. Oxygen exchange experiments were also performed in order to allow calculation of the absolute rate constant of *E-P hydrolysis to the noncovalent complex (0.8-1.0 s-1), which differs from the observed rate of enzyme dephosphorylation (0.3-0.5 s-1); in addition, they allowed calculation of the bimolecular rate constant of substrate binding (2-2.4 M-1 s-1). The results demonstrate that in the presence of dimethyl sulfoxide, phosphorylation occurs by the following simple mechanism: relatively slow binding of the neutral substrate (Mg X Pi), with poor affinity, followed by a thermodynamically favorable formation of the covalent bond between phosphate and the possibly hydrophobic active site. The interaction between magnesium and calcium-deprived SR vesicles was studied in the presence of 0-20% v/v dimethyl sulfoxide (or 0-30% v/v glycerol) at pH 7 and 20 degrees C. The presence of either solvent led to the disappearance of the two typical pH-dependent effects we previously characterized for magnesium: loss of the Mg2+-induced spectral shift of tryptophan fluorescence emission and loss of the biphasic pattern displayed by the intrinsic fluorescence rise after addition of calcium to Ca2+-deprived Mg2+-preincubated vesicles. In the absence of solvent, the interaction of magnesium with the calcium-deprived ATPase was also characterized from the point of view of phosphoenzyme formation from ATP or Pi at pH 7 in the absence of potassium: we found that calcium-independent phosphorylation was slower when phosphate was added to SR vesicles preincubated with magnesium that when magnesium was added to vesicles preincubated with phosphate, suggesting that preincubation with magnesium had depleted the phosphate-reactive conformation of the ATPase. A simple reaction scheme for phosphoenzyme formation is described: it implies that the (Mg X Pi) complex is a substrate for this reaction, whereas the Mg2+ itself acts as a pH-dependent, dimethyl sulfoxide sensitive inhibitor of full enzyme phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-1 inhibits the synthesis of collagen by fibroblasts

Human dermal fibroblasts, exposed to human or porcine Interleukin-1, responded by an inhibition of collagen synthesis in a dose dependent manner. Incubation with Il-1 for more than 8 h was required to see an appreciable effect. The phenomenon was not dependent on the presence of serum in the culture medium. Since a stimulation of prostaglandin E2 secretion was also observed in presence of Il-1, we investigated the eventual role of arachidonic acid metabolites in the phenomenon. Inhibitors interfering with arachidonate metabolism, namely indomethacin, acetyl salicylic acid, BW 755 C and NDGA had no influence on the inhibition of collagen synthesis caused by Il-1. These data suggest that both cyclooxygenase and lipoxygenase derived metabolites of arachidonic acid are unlikely to play a role in the mechanism.