Apoflavodoxin folding mechanism: an alpha/beta protein with an essentially off-pathway intermediate.

The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient intermediate accumulates during the folding reaction. The intermediate is monomeric, and it is not related to proline isomerization. Unlike many cases where the presence of an intermediate has been detected either by a burst phase or by the curvature, at low urea concentration, of the otherwise only observable kinetic phase, two kinetic phases are observed in apoflavodoxin folding whose total amplitude equals the amplitude of unfolding. To determine the role of the intermediate in the folding reaction, the apoflavodoxin kinetic data have been fitted to all conceivable three-species kinetic models (either linear or triangular). Using a stepwise fitting procedure, we find that the off-pathway mechanism explains most of the kinetic data (not a slow unfolding phase), the on-pathway mechanism being rejected. By using global analysis, good overall agreement between data and fit is found when a triangular mechanism is considered. The fitted values of the microscopic constants indicate that most of the unfolded molecules refold from the denatured state. Apoflavodoxin thus folds via a triangular, but essentially off-pathway, mechanism. We calculate that the retardation of the folding caused by the off-pathway intermediate is not large. Some unusual properties of the intermediate are discussed.     

Histidine residues at the N- and C-termini of alpha-helices: perturbed pKas and protein stability.

A single histidine residue has been placed at either the N-terminus or the C-terminus of each of the two alpha-helices of barnase. The pKa of that histidine residue in each of the four mutants has been determined by 1H NMR. The pKas of the two residues at the C-terminus are, on average, 0.5 unit higher, and those of the residues at the N-terminus are 0.8 unit lower, than the pKa of histidines in unfolded barnase at low ionic strength. The conformational stability of the mutant proteins at different values of pH has been measured by urea denaturation. C-Terminal histidine mutants are approximately 0.6 kcal mol-1 more stable when the introduced histidine is protonated, both at low and high ionic strength. N-Terminal mutants with a protonated histidine residue are approximately 1.1 kcal mol-1 less stable at low ionic strength and 0.5 kcal mol-1 less stable at high ionic strength (1 M NaCl). The low-field 1H NMR spectra of the mutant proteins at low pH suggest that the C-terminal histidines form hydrogen bonds with the protein while the N-terminal histidines do not form the same. The perturbations of pKa and stability result from a combination of different electrostatic environments and hydrogen-bonding patterns at either ends of helices. The value of 0.6 kcal mol-1 represents a lower limit to the favorable electrostatic interaction between the alpha-helix dipole and a protonated histidine residue at the C-terminal end of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissection of an enzyme by protein engineering. The N and C-terminal fragments of barnase form a native-like complex with restored enzymic activity.

A method is described for producing fragments of a protein suitable for studies of protein folding. The codon for a single methionine residue is introduced into the cloned gene of barnase, and the gene product cleaved with cyanogen bromide. The site of mutation was chosen to be at the surface of the protein in a region connecting segments of secondary structure in the native enzyme. The alpha + beta protein was mutated from Val36----Met, and split into two fragments, B(1-36) containing the alpha-helical regions and B(37-110), the beta-sheet. The fragments were purified by ion exchange chromatography. Neither retains catalytic activity. Fluorescence, circular dichroism, and 1H nuclear magnetic resonance data indicate that their structures are each close to that of random-coil peptides. The two fragments associate to form a tight complex (Kd = 0.2 to 0.6 microM), which displays spectroscopic properties similar to those of the uncleaved protein. The catalytic activity is restored in the complex with a value for Km similar to that for native enzyme but with kcat reduced about three- to fourfold. The second-order rate constant for association on mixing fragments in the concentration range 2.5 to 7.5 microM is 1 x 10(5) s-1 M-1.     

The influences of calcium and magnesium ions on the exchange of monovalent cations in Neurospora crassa

Low-K⁺, high-Na⁺ cells of strain RL21a of Neurospora crassa , in steady state with 25 m M Na⁺, were used to study K⁺/Na⁺ exchanges in the presence or absence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺ and Mg²⁺, a low concentration of K⁺ (0.3 m M ) triggered a rapid exchange, but in the absence of the divalents, a high K⁺ concentration (30 m M ) was required to initiate the exchange at a rapid rate. In the absence of Ca²⁺ and Mg²⁺, K⁺ uptake did not occur at low K⁺ concentration, internal K⁺ did not regulate Na⁺ influx in the presence of external K⁺, and the efflux of Na⁺ proceeded at maximum activity at very low-K⁺ contents.     

CD38 and CD157 as receptors of the immune system: a bridge between innate and adaptive immunity

This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage.     

Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.     

Intrinsic caspase-8 activation mediates sensitization of erlotinib-resistant tumor cells to erlotinib/cell-cycle inhibitors combination treatment

Inhibitors of the tyrosine kinase activity of epidermal growth factor receptor, as erlotinib, have an established role in treating several cancer types. However, resistance to erlotinib, particularly in breast cancer cell lines, and erlotinib treatment-associated disorders have also been described. Also, methods and combination therapies that could reverse resistance and ameliorate non-desirable effects represent a clinical challenge. Here, we show that the ATP non-competitive CDK2/cyclin A inhibitor NBI1 sensitizes erlotinib-resistant tumor cells to the combination treatment (co-treatment) for apoptosis-mediated cell death. Furthermore, in erlotinib-sensitive cells, the effective dose of erlotinib was lower in the presence of NBI1. The analysis in the breast cancer MDA-MB-468 erlotinib-resistant and in lung cancer A549 cell lines of the molecular mechanism underlying the apoptosis induced by co-treatment highlighted that the accumulation of DNA defects and depletion of cIAP and XIAP activates the ripoptosome that ultimately activates caspases-8 and -10 and apoptosis. This finding could have significant implications for future treatment strategies in clinical settings.     

Histidine-aromatic interactions in barnase. Elevation of histidine pKa and contribution to protein stability.

Aromatic side-chains are found in the vicinity of histidine residues in many proteins and protein complexes. We have studied the interaction between a histidine residue (His18) and aromatic residues at position 94 in barnase. Three different techniques have been applied to show that Trp94 interacts more strongly with the protonated form of His18. The aromatic-histidine interaction stabilizes the protonated form of histidine by 0.8 to 1 kcal mol-1 relative to the unprotonated and, thereby, increases its pKa value. This was shown indirectly from the pH dependence of the stability of the wild-type protein and the mutant Trp94----Leu; and directly from the difference in pKa of His18 between wild-type barnase and the same mutant protein, and from double-mutant cycles that measure the total interaction energy of Trp94 with His18 at both low and high pH. When Trp94 is replaced by other aromatic amino acids, the strength of the interaction decreases in the series His-Trp greater than His-Tyr greater than His-Phe. The interaction is not masked by high salt concentrations. The raising of the pKa value of His18 by interaction with Trp94 is shown to be consistent with solution studies with model compounds. The histidine-aromatic interaction could have implications in binding and catalysis for modulation of the histidine pKa value.     

An early effect of estradiol at hepatic level, previous to its protein synthesis activation

Liver glycogen phosphorylase activity is increased before protein synthesis activation by estradiol. This effect is not blocked by antibiotics (actinomycin D and cycloheximide) inhibitor of protein synthesis. At times very similar to those of phosphorylase activation, cAMP levels are not enhanced, as would be expected, but slightly depleted. At similar times, cGMP levels are dramatically increased.