Differential effect of cytokine treatment on Fc alpha receptor I- and Fc gamma receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages

Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (Fc gamma RI) and the IgA FcR (Fc alpha RI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-gamma, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to Fc alpha RI or Fc gamma RI on MDM. Although Fc alpha RI and Fc gamma RI share a common signaling pathway contingent on association with the gamma-chain (FcR gamma subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by Fc alpha RI and Fc gamma RI; however, IFN-gamma-treated MDM phagocytosed tumor cells only with the Fc gamma RI-directed bispecific Abs. Similarly, IFN-gamma-cultured MDM lysed tumor cells more efficiently via Fc gamma RI then by Fc alpha RI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via Fc alpha RI than Fc gamma RI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-gamma-mediated enhancement of Fc gamma RI expression and Fc gamma RI gamma-chain complexes, the regulation of Fc gamma RI- or Fc alpha RI-mediated activity occurred without significant change in either receptor expression or total complexes with gamma subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.     

On the temperature-induced coil to globule transition of poly-N-isopropylacrylamide in dilute aqueous solutions

Poly-N-isopropylacrylamide (PNIPAM) is a chemical isomer of poly-leucine, having the polar peptide group in the side-chain rather than in the backbone. It has been demonstrated experimentally that PNIPAM dissolved in aqueous solution undergoes a collapse transition from coil to globule on increasing temperature above the θ-point. By a careful reviewing of existing experimental data, we emphasize that such coil to globule collapse has to be considered an intramolecular first-order transition, analogous to the cold renaturation of small globular proteins. The main theoretical approaches to the coil to globule collapse in homopolymers are discussed briefly, and a critical comparison between the existing models is performed. We point out that, as a general result, the coil to globule collapse is expected to be a first-order transition for rigid and semi-rigid macromolecules. Finally, taking advantage of the analogy between the coil to globule collapse of PNIPAM and the cold renaturation of small globular proteins, we try to clarify some important and intriguing aspects of protein thermodynamics. This leads to the conclusion that the amphiphilic nature of polypeptide chain plays the fundamental role for the existence of two temperature-induced conformational transitions.     

Metabolic responses in cucumber (Cucumis sativus L.) roots under Fe-deficiency: a 31P-nuclear magnetic resonance in-vivo study

The metabolic responses occurring in cucumber (Cucumis sativus L.) roots (a strategy-I plant) grown under iron-deficiency conditions were studied in-vivo using ³¹P-nuclear magnetic resonance spectroscopy. Iron starvation induced activation of metabolism leading to the consumption of stored carbohydrates to produce the NAD(P)H, ATP and phosphoenolpyruvate necessary to sustain the increased activity of the NAD(P)H:Fe³⁺-reductase, the H⁺-ATPase (EC 3.6.1.35) and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Activation of catabolic pathways was supported by the enhancement of glycolytic enzymes and concentrations of the metabolites glucose-6-phosphate and fructose-6-phosphate, and by enhancement of the respiration rate. Moreover, Fe-deficiency induced a slight increase in the cytoplasmic (pH_c) and vacuolar (pH_v) pHs as well as a dramatic decrease in the vacuolar phosphate (Pi) concentration. A comparison was done using fusicoccin (FC), a fungal toxin which stimulates proton extrusion. Changes in pH_c and pH_v were measured after addition of FC. Under these conditions, a dramatic alkalinization of the pH_v of −Fe roots was observed, as well as a concomitant Pi movement from the vacuole to the cytoplasm. These results showed that Fe starvation was indeed accompanied by the activation of metabolic processes useful for sustaining the typical responses occurring at the plasma-membrane level (i.e. increases in the NAD(P)H:Fe³⁺-reductase and H⁺-ATPase activities) as well as those involved in the homeostasis of pH_c. The decrease in vacuolar Pi levels induced by Fe-deficiency and FC and movement of Pi from the vacuole to the cytoplasm suggest a possible involvement of this compound in the cellular pH-stat system. Received: 30 July 1999 / Accepted: 11 November 1999     

Role of BAG3 protein in leukemia cell survival and response to therapy

The ability of BAG3, a member of the BAG family of heat shock protein (Hsp) 70 - cochaperones, to sustain the survival of human primary B-CLL and ALL cells was recognized about nine years ago. Since then, the anti-apoptotic activity of BAG3 has been confirmed in other tumor types, where it has been shown to regulate the intracellular concentration and localization of apoptosis-regulating factors, including NF-κB-activating (IKKγ) and Bcl2-family (Bax) proteins. Furthermore, growing evidences support its role in lymphoid and myeloid leukemia response to therapy. Moreover in the last years, the contribution of BAG3 to autophagy, a process known to be involved in the pathogenesis and response to therapy of leukemia cells, has been disclosed, opening a new avenue for the interpretation of the role of this protein in leukemias' biology.     

Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.     

Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.     

Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.     

Protein thiolation index (PTI) as a biomarker of oxidative stress

Several biomarkers of oxidative stress have been proposed and used in clinical research but so far unreliable or, at least, controversial results have been obtained. Given the high susceptibility of sulfhydryl groups to oxidation, we here suggest the use of a protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to plasma proteins (forming, as a whole, S-thiolated proteins) and protein free cysteinyl residues, as a suitable biomarker of oxidative stress. While titration of free thiols can be performed by a simple spectrophotometric procedure, accurate quantification of S-thiolated proteins is problematic and current methods require, in most cases, application of time-consuming chromatographic techniques, making their application to large-scale clinical studies difficult. Here we report a new spectrophotometric method which relies on the specific determination of low molecular mass thiols released from S-thiolated proteins after dithiothreitol reduction. These amino acids can be titrated by conjugation with ninhydrin which, reacting with primary and secondary amine groups, yields a deep blue-purple color, which can be spectrophotometrically revealed. PTI showed an age dependency with a near linear increase during aging in humans. In addition, PTI was significantly higher in patients suffering from alkaptonuria with respect to healthy controls, suggesting that increased prooxidant conditions occur in the blood of these subjects.