YAP/TAZ mechano-transduction as the underlying mechanism of neuronal differentiation induced by reduced graphene oxide.

AIM: The aim of this work is the dissection of the molecular pathways underlying the differentiation effect of reduced graphene oxide (GO) materials in the absence of differentiation agents. MATERIALS & METHODS: Reduced GO is obtained either by drop casting method and heat-treated or biological reduction by the interaction between GO and wtPrxI. Cells were grown on both materials and the differentiation process studied by immunological and morphological detection. RESULTS & CONCLUSION: The results obtained indicate that both reduction methods of GO can determine the modulation of pathway involved in mechano-transduction and differentiation, by affecting YAP/TAZ localization outside the nuclei and increasing neuronal differentiation markers. This suggests that the mechano-transduction pathways are responsible for the differentiation process.

N6L pseudopeptide interferes with nucleophosmin protein-protein interactions and sensitizes leukemic cells to chemotherapy.

NPM1 is a multifunctional nucleolar protein implicated in several processes such as ribosome maturation and export, DNA damage response and apoptotic response to stress stimuli. The NPM1 gene is involved in human tumorigenesis and is found mutated in one third of acute myeloid leukemia patients, leading to the aberrant cytoplasmic localization of NPM1. Recent studies indicated that the N6L multivalent pseudopeptide, a synthetic ligand of cell-surface nucleolin, is also able to bind NPM1 with high affinity. N6L inhibits cell growth with different mechanisms and represents a good candidate as a novel anticancer drug for a number of malignancies of different histological origin. In this study we investigated whether N6L treatment could drive antitumor effect in acute myeloid leukemia cell lines. We found that N6L binds NPM1 at the N-terminal domain, co-localizes with cytoplasmic, mutated NPM1, and interferes with its protein-protein associations. N6L toxicity appears to be p53 dependent but interestingly, the leukemic cell line harbouring the mutated form of NPM1 is more resistant to treatment, suggesting that NPM1 cytoplasmic delocalization confers protection from p53 activation. Moreover, we show that N6L sensitizes AML cells to doxorubicin and cytarabine treatment. These studies suggest that N6L may be a promising option in combination therapies for acute myeloid leukemia treatment.

PPARs in Neurodegenerative and Neuroinflammatory Pathways.

BACKGROUND: PPARs are lipid sensors activated by dietary lipids or their metabolites, mainly fatty acids and eicosanoids, that play critical roles in CNS biology, since brain has a very high lipid content and has the higher energetic metabolism in the body. METHODS: In neurodegenerative diseases in addition to metabolic impairment, also neuroinflammation is observed and PPARs are also closely linked to inflammatory processes. Several studies have revealed a complicated relationship between the innate immune response and tissue metabolism. RESULTS: In the brain, during pathological conditions, an alteration in metabolic status occurs, particularly involving glucose utilization and production, a condition which is generally related to metabolic changes. CONCLUSION: Taking into account the high expression of PPARs in the brain, this review will focus on the role of these transcription factors in CNS diseases.

Roles of PPAR transcription factors in the energetic metabolic switch occurring during adult neurogenesis.

PPARs are a class of ligand-activated transcription factors belonging to the superfamily of receptors for steroid and thyroid hormones, retinoids and vitamin D that control the expression of a large number of genes involved in lipid and carbohydrate metabolism and in the regulation of cell proliferation, differentiation and death. The role of PPARs in the CNS has been primarily associated with lipid and glucose metabolism; however, these receptors are also implicated in neural cell differentiation and death, as well as neuronal maturation. Although it has been demonstrated that PPARs play important roles in determining NSCs fate, less is known about their function in regulating NSCs metabolism during differentiation. In order to identify the metabolic events, controlled by PPARs, occurring during neuronal precursor differentiation, the glucose and lipid metabolism was followed in a recognized model of neuronal differentiation in vitro, the SH-SY5Y neuroblastoma cell line. Moreover, PPARs distribution were also followed in situ in adult mouse brains. The concept of adult neurogenesis becomes relevant especially in view of those disorders in which a loss of neurons is described, such as Alzheimer disease, Parkinson disease, brain injuries and other neurological disorders. Elucidating the crucial steps in energetic metabolism and the involvement of PPARγ in NSC neuronal fate (lineage) may be useful for the future design of preventive and/or therapeutic interventions.

Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis.

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.

Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways.

The effect of AZT and chloroquine on the activities of ricin and a saporin-transferrin chimeric toxin.

This study deals with the combination of chloroquine (CQ, an anti-malaric drug) and 3'-azido-3'-deoxythymidine (AZT, anti-human immuno-deficiency virus (HIV) drug) with a chimeric toxin (TS) obtained by chemical linking of saporin (a ribosome inactivating protein from the plant Saponaria officinalis) and human transferrin, in the intoxication of the human chronic myeloid leukaemia cells (K562). Our data demonstrate that AZT, at concentrations comparable to those reached in the blood of HIV-infected patients under pharmacological treatment with this drug, can increase the toxicity of TS in cooperation with CQ inducing an increased effect on protein synthesis in K562 cells ( approximately 50% inhibition of protein synthesis for TS alone, and TS with AZT and approximately 70% with both AZT and CQ). Furthermore, pre-treatment of cells with AZT alone can induce an increase of apoptosis in K562 cells intoxicated with TS. By comparing data obtained with the model toxin ricin, we get indications that the two toxins partially differ in their intracellular routes, also suggesting that chimeric constructs containing ricin-like toxins (i.e. immunotoxins) could be coupled with the use of common and cheap drugs for the treatment of cancer in HIV-infected patients.

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy.

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM). The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome.

The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy.

With the electro-driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer-aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady-state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic-to-measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20-fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.

The effect of monensin and chloroquine on the endocytosis and toxicity of chimeric toxins.

The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e., endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell.

Crystallization and preliminary X-ray study of saporin, a ribosome-inactivating protein from Saponaria officinalis.

Single crystals of the protein saporin isolated from the seeds of S. officinalis have been grown by the vapor-diffusion method using ammonium sulfate as precipitant. The crystals are tetragonal, space group P4122 (P4322), with cell dimensions a = b = 67.53 and c = 119. 67 A, and diffract to 2.0 A resolution on a rotating-anode X-ray source. The asymmetric unit contains one molecule, corresponding to a volume of the asymmetric unit per unit mass (Vm) of 2.38 A3 Da-1.

Targeting of saporin to Hodgkin's lymphoma cells by anti-CD30 and anti-CD25 bispecific antibodies.

CD25 and CD30 represent suitable target molecules for bispecific antibody (bimAb)-driven toxin delivery to lymphoid tumour cells. We describe two new anti-CD30/anti-saporin bimAbs (termed CD30 x sap1 and CD30 x sap2), produced by hybrid hybridomas, which react against non-cross-reactive epitopes of the saporin molecule, and compared their effect with a bimAb reacting with saporin and with CD25 (CD25 x sap1). In a protein synthesis inhibition assay these bimAbs were able to enhance saporin toxicity (IC50 = 8.5 x 10(-9) M in the absence of mAbs) with a similar activity: in the presence of 10(-9) M CD30 x sap1 bimAb the IC50 was 2.75 x 10(-11) M, whereas with CD30 x sap2 bimAb the IC50 was 6.5 x 10(-11) M and CD25 x sap1 bimAb displayed an IC50 of 3 x 10(-11) M (as saporin). The combined use of the two anti-CD30 bimAbs further increased cytotoxicity by 100-fold, resulting in an IC50 of 1.9 x 10(-13) M. A slightly less efficient improvement was obtained by combining the CD25 x sap1 bimAb with the CD30 x sap2 bimAb directed against a different toxin epitope (saporin IC50 to 7 x 10(-13) M). In contrast, no synergistic effect was observed using the combination of the anti-CD25 bimAb with the anti-CD30 bimAb reacting with the same epitope of saporin (IC50 = 4.5 x 10(-11) M). Analysis of FITC-saporin binding to L540 cells by flow cytometry demonstrated that the appropriate combinations of the two anti-CD30/anti-saporin bimAbs or of the anti-CD30/anti-saporin and anti-CD25/anti-saporin bimAbs had a cooperative effect on the binding of the ribosome-inactivating protein (RIP) to the cells, when compared with single bimAbs.

Covalent complex of microperoxidase with a 21-residue synthetic peptide as a maquette for low-molecular-mass redox proteins.

Here we report the structural and functional characterization of a covalent complex (MKP) obtained by cross-linking microperoxidase (Mp), the haem-undecapeptide obtained by the peptic digestion of cytochrome c, with a 21-residue synthetic peptide (P21) analogous to the S-peptide of the RNase A. The covalent complex has been prepared by introducing a disulphide bond between Cys-1 of P21 and Lys-13 of Mp, previously modified with a thiol-containing reagent. On formation of the complex (which is a monomer), the helical content of P21 increases significantly. The results obtained indicate that His-13 of P21 co-ordinates to the sixth co-ordination position of the haem iron, thus leading to the formation of a complex characterized by an equilibrium between an 'open' and a 'closed' structure, as confirmed by molecular dynamics simulations. Under acidic pH conditions, where His-13 of P21 is loosely bound to the haem iron ('open' conformation), MKP displays appreciable, quasi-reversible electrochemical activity; in contrast, at neutral pH ('closed' conformation) electrochemical behaviour is negligible, indicating that P21 interferes with the electron-transfer properties typical of Mp. On the whole, MKP is a suitable starting material for building a miniature haem system, with interesting potential for application to biosensor technology.

Liposomal targeting of leukaemia HL60 cells induced by transferrin-receptor endocytosis.

A liposomal carrier system able to interact specifically with HL60 leukaemia cells was produced using small unilamellar liposomes made of pure phospholipids chemically cross-linked to human transferrin. The conjugation of transferrin to liposomes was carried out using N-succinimidyl 3-(2-pyridyldithio)-propionate and 2-iminothiolane as activating agents for the liposomes and the protein. The reaction occurred under conditions set to covalently link on the surface of a single vesicle a limited number (one to ten) of transferrin molecules, as verified by means of electron microscopy and immunoenzymic measurements. Before conjugation, the ultrastructure of the liposomes, and the content and distribution of the amino groups within the bilayer, were determined. The reactivity of the liposomes towards amino-derivatizing or thiolating compounds was also measured. Kinetic spectroscopic measurements confirmed that the distribution of the phosphatidylethanolamine in the vesicle bilayer is asymmetrical: 22% of phosphatidylethanolamine was found exposed to the external surface of the liposomes and accessible to the cross-linker. The modified liposomes were able to interact specifically with the cells and to be internalized by active receptor-mediated endocytosis, as demonstrated by the full inhibition of internalization induced by free transferrin.

A saporin-insulin conjugate: synthesis and biochemical characterization.

Saporin, a single-chain, non-cytotoxic, ribosome-inactivating protein from Saponaria officinalis, was chemically linked to the hormone insulin in a 1:1 complex. To follow by dynamic video microscopy the endocytosis and intracellular transport in vivo, a second covalent conjugate with a saporin derivative labelled with fluorescein isothiocyanate was also prepared. Both conjugates were characterized with reference to homogeneity, stoichiometry, optical spectroscopy and toxicity. Both were found to exhibit scarce toxicity toward both CHO and HEP G2 cells; optical video microscopy on living cells indicates that reduced toxicity may be (partly) due to a very limited binding of the saporin-insulin conjugate to membrane receptors. These results suggest a strategy for new possible covalent conjugates of saporin with alternative and specific macromolecular carriers.

A chimeric saporin-transferrin conjugate compared to ricin toxin: role of the carrier in intracellular transport and toxicity.

Human transferrin (Tf) and saporin-6 (Sap), a ribosome inactivating protein from Saponaria officinalis, were chemically conjugated: the reaction generated two chimeras (called Tf-Sap) that proved to be cytotoxic to HepG2 cells. Electrophoretic and chromatographic analysis revealed that the two conjugates contained saporin and Tf in a 2:1 or 1:1 molar ratio (140 and 110 KDa, respectively). Free saporin is essentially nontoxic, whereas Tf-Sap efficiently kills HepG2 cells, although its ID50 (= 6 nM) is 1000-fold greater than that of ricin. Intracellular transport of these toxins was followed by in vivo fluorescence video microscopy, preparing the conjugates starting from rhodamine isothiocyanate-labeled saporin. Image analysis of living HepG2 cells exposed to fluorescent Tf-Sap revealed that the endocytotic pathway involving passage through secondary endosomes is dictated by Tf and is different from that of ricin (the dimeric toxin from Ricinus communis), which is delivered to the Golgi apparatus, the probable site of activation. We discuss whether differences in toxicity between ricin and Tf-Sap can be attributed to the different mechanisms of transport and activation.

Analysis of biochemical processes in single living cells by quantitative microscopy.

Spectroscopic measurements in single living cells are made possible by the development of computer controlled light detectors, which, when applied to optical microscopes, yield spatial, temporal and eventually spectroscopic information about the sample. This minireview describes some experiments in which the distribution and concentration of specific intracellular markers (proteins, protein complexes, RNA) has been followed by quantitative microscopy. The examples chosen have contributed to shed light on a biochemical process as it happens in vivo; because of the non ideal conditions of the intracellular milieu, the comparison of in vivo and in vitro experiments is of great relevance to the understanding of cellular physiology.

Intracellular dynamics of ricin followed by fluorescence microscopy on living cells reveals a rapid accumulation of the dimeric toxin in the Golgi apparatus.

The intracellular dynamics of fluorescent conjugates of the toxic lectin ricin was followed by video fluorescence microscopy on living CHO cells, demonstrating that the ricin heterodimer and its isolated B chain, after binding to the plasma membrane receptors, migrate to and accumulate in the Golgi apparatus following internalization. A ricin derivative labelled with fluorescein on the A chain and rhodamine on the B chain did not display significant splitting of the A-B heterodimer during translocation of the toxin to the Golgi; this novel finding provides support for the hypothesis that further processing of ricin takes place in this cellular compartment.