Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.

The mechanism of Hsp70 chaperones: (entropic) pulling the models together.

Hsp70s are conserved molecular chaperones that can prevent protein aggregation, actively unfold, solubilize aggregates, pull translocating proteins across membranes and remodel native proteins complexes. Disparate mechanisms have been proposed for the various modes of Hsp70 action: passive prevention of aggregation by kinetic partitioning, peptide-bond isomerase, Brownian ratcheting or active power-stroke pulling. Recently, we put forward a unifying mechanism named 'entropic pulling', which proposed that Hsp70 uses the energy of ATP hydrolysis to recruit a force of entropic origin to locally unfold aggregates or pull proteins across membranes. The entropic pulling mechanism reproduces the expected phenomenology that inspired the other disparate mechanisms and is, moreover, simple.

Inhibition of the renin-angiotensin system does not reduce platelet activity at rest or during stress in hypertension.

OBJECTIVES: We investigated the influence of angiotensin receptor blockade and angiotensin-converting enzyme inhibition on stress-induced platelet activation in hypertensive patients. Secondary aims were effects on inflammation, coagulation, and endothelial function. METHODS: Following a 4-week placebo period, 25 hypertensive patients entered a double-blind, crossover study comparing enalapril (20 mg once daily) and losartan (100 mg once daily) treatment (each for 8 weeks). Patients were studied at rest and after a standardized exercise test. RESULTS: Mean arterial pressure was reduced from 119 ± 2 to 104 ± 2 (enalapril) and 106 ± 2 (losartan) mmHg (both P <0.001). Plasma angiotensin II decreased from 2.4 ± 0.4 to 0.5 ± 0.1 pmol/l with enalapril, and increased to 7.2 ± 1.3 pmol/l with losartan (both P <0.001). Exercise-evoked platelet activation, as evidenced by increased numbers of P-selectin-positive platelets (P <0.01), elevated circulating platelet-platelet aggregates (P <0.01) and soluble P-selectin levels (P <0.001), and increased platelet responsiveness to adenosine diphosphate and thrombin (both P <0.05). Neither drug influenced these markers of platelet activation at rest or following exercise. Markers of inflammation (high-sensitivity C reactive protein, interleukin-6, tissue necrosis factor-α), coagulation (tissue plasminogen activator antigen, prothrombin fragment F1+2), and endothelial function (von Willebrand factor, soluble vascular cellular adhesion molecule-1, and intercellular adhesion molecule-1) were also uninfluenced by treatment. CONCLUSION: Enalapril and losartan failed to reduce platelet activity both at rest and during exercise in hypertensive patients. Markers of inflammation, coagulation, and endothelial function were similarly unaffected. Inhibition of the renin-angiotensin system promotes its beneficial effects in hypertension through mechanisms other than platelet inhibition.

Long-lasting antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of glucagon-like peptide-1.

Glucagon-like peptide-1(7-37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 +/- 0.14 and 1.39 +/- 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 +/- 0.05 and 0.36 +/- 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.

The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin.

Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P). G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P. G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution. G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P. Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture. The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin. Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate. G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex. We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication.

Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

Extrasynaptic neurotransmission in the modulation of brain function. Focus on the striatal neuronal glial networks

Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT) and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR) heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT) and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT) and histamine striatal afferents, the cholinergic interneurons, and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal cellular networks

Structure and function of RecA-DNA complexes.

While the E. coli RecA protein has been the most intensively studied enzyme of homologous recombination, the unusual RecA-DNA filament has stood alone until very recently. It now appears that this protein is part of a universal family that spans all of biology, and the filament that is formed by the protein on DNA is a universal structure. With RecA's role in recombination given new and greatly increased significance, we focus in this review on the energetics of the RecA-mediated strand exchange and the relation between the energetics and recombination spanning heterologous inserts.

Novel Bioconjugates of Aminolevulinic Acid with Nucleosides

Esters and amino acid derivatives of 5-aminolevulinic acid (ALA) are efficient prodrugs for the production of protoporphyrin IX (PpIX), which has been used in photodynamic cancer therapy (PDT). The synthesis of novel bioconjugates combining ALA with adenosine and thymidine is reported. The novel bioconjugates have been made using a robust methodology. The new class of prodrugs contains one, two, or three ALA per molecule. Preliminary cell tests in human cancer cell lines indicate that the thymidine conjugate of ALA is an efficient prodrug for PDT.

Transient suppression of Shigella flexneri type 3 secretion by a protective O-antigen-specific monoclonal IgA.

Mucosal immunity to the enteric pathogen Shigella flexneri is mediated by secretory IgA (S-IgA) antibodies directed against the O-antigen (O-Ag) side chain of lipopolysaccharide. While secretory antibodies against the O-Ag are known to prevent bacterial invasion of the intestinal epithelium, the mechanisms by which this occurs are not fully understood. In this study, we report that the binding of a murine monoclonal IgA (IgAC5) to the O-Ag of S. flexneri serotype 5a suppresses activity of the type 3 secretion (T3S) system, which is necessary for S. flexneri to gain entry into intestinal epithelial cells. IgAC5's effects on the T3S were rapid (5 to 15 min) and were coincident with a partial reduction in the bacterial membrane potential and a decrease in intracellular ATP levels. Activity of the T3S system returned to normal levels 45 to 90 min following antibody treatment, demonstrating that IgAC5's effects were transient. Nonetheless, these data suggest a model in which the association of IgA with the O-Ag of S. flexneri partially de-energizes the T3S system and temporarily renders the bacterium incapable of invading intestinal epithelial cells. IMPORTANCE: Secretory IgA (S-IgA) serves as the first line of defense against enteric infections. However, despite its well-recognized role in mucosal immunity, relatively little is known at the molecular level about how this class of antibody functions to prevent pathogenic bacteria from penetrating the epithelial barrier. It is generally assumed that S-IgA functions primarily by "immune exclusion," a phenomenon in which the antibody binds to microbial surface antigens and thereby promotes bacterial agglutination, entrapment in mucus, and physical clearance from the gastrointestinal tract via peristalsis. The results of the present study suggest that in addition to serving as a physical barrier, S-IgA may have a direct impact on the ability of microbial pathogens to secrete virulence factors required for invasion of intestinal epithelial cells.

Probing Rad51-DNA interactions by changing DNA twist.

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.