BBA - Molecular Cell Research (v.1763, #1)

Sleeping sickness: PEX and drugs by Wolfgang Schliebs (4-5).
Finding new ways in the treatment of fatal parasitic diseases like the human sleeping sickness is a major challenge of biomedical research. The growing body of knowledge about the biogenesis of the glycosome, a peroxisome-related organelle of trypanosomes, might allow defining novel targets for drug development.
Keywords: Glycosome; Peroxisome; Biogenesis; Trypanosoma;

Protozoan Kinetoplastida such as the pathogenic trypanosomes compartmentalize several important metabolic systems, including the glycolytic pathway, in peroxisome-like organelles designated glycosomes. Genes for three proteins involved in glycosome biogenesis of Trypanosoma brucei were identified. A preliminary analysis of these proteins, the peroxins PEX6, PEX10 and PEX12, was performed. Cellular depletion of these peroxins by RNA interference affected growth of both mammalian bloodstream-form and insect-form (procyclic) trypanosomes. The bloodstream forms, which rely entirely on glycolysis for their ATP supply, were more rapidly killed. Both by immunofluorescence studies of intact procyclic T. brucei cells and subcellular fractionation experiments involving differential permeabilization of plasma and organellar membranes it was shown that RNAi-dependent knockdown of the expression of each of these peroxins resulted in the partial mis-localization of different types of glycosomal matrix enzymes to the cytoplasm: proteins with consensus motifs such as the C-terminal type 1 peroxisomal targeting signal PTS1 or the N-terminal signal PTS2 and a protein for which the sorting information is present in a polypeptide-internal fragment not containing an identifiable consensus sequence.
Keywords: Trypanosoma; Glycosome biogenesis; Peroxin; RNA interference;

Adrenergic modulation of the type 1 IP3 receptors in the rat heart by Dana Jurkovicova; Lucia Kubovcakova; Sona Hudecova; Richard Kvetnansky; Olga Krizanova (18-24).
Inositol 1,4,5-trisphosphate (IP3) receptors are calcium-releasing channels localized on the sarcoplasmic reticulum. IP3 receptors mediate the calcium mobilizing effect of a wide range of hormones, cytokines, and neurotransmitters and play an important role in variety of cell functions. The aim of this work was to study, how partial depletion of catecholamines affects the gene expression and protein levels of the type 1 IP3 receptors in rat heart. The type 1 IP3 receptor mRNA levels were studied in the left cardiac atrium and ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. The 6-OHDA produces anatomical and functional denervation resulting in decreased levels of noradrenaline and adrenaline. We also used corticoliberin (CRH) knockout mice, where secretion of adrenaline is significantly suppressed. Administration of 6-OHDA significantly decreases mRNA levels of the type 1 IP3 receptor in both, the left atrium and the left ventricle, while the gene expression of the sarcoplasmic reticular Ca2+-ATPase (SERCA 2) was unaffected. CRH knockout mice possess markedly lower levels of the type 1 IP3 receptor mRNA compared to wild-type mice in both, control and stressed conditions. These data point to the adrenergic modulation of the type 1 IP3 receptors in the rat hearts.
Keywords: Type 1 IP3 receptor; 6-hydroxydopamine; Cardiac atria; Cardiac ventricle; CRH knockout mouse; Catecholamine;

Protein kinase C-δ (PKC-δ) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-δ inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCδ activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-δ activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 μM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-δ in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125FAK TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125FAK TyrP, MAPK activation and PKC-δ TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-δ blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-δ in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-δ investigation.
Keywords: Cholecystokinin; Protein kinase C; Pancreatic acini; Exocrine secretion; PKC inhibitors; Rottlerin;

The telomerase inhibitor telomestatin induces telomere shortening and cell death in Arabidopsis by Lili Zhang; Katsunori Tamura; Kazuo Shin-ya; Hideo Takahashi (39-44).
The cellular response to telomere dysfunction in plants was investigated with the use of telomestatin, an inhibitor of human telomerase activity. Telomestatin bound to plant telomeric repeat sequence, and inhibited telomerase activity in suspension-cultured cells of Arabidopsis thaliana and Oryza sativa (rice) in a dose-dependent manner. The inhibitor did not affect transcript level of the TERT gene, which encodes the catalytic subunit of telomerase, in the plant cells. Inhibition of telomerase activity by telomestatin resulted in rapid shortening of telomeres and the induction of cell death by an apoptosis-like mechanism in Arabidopsis cells. These results suggest that telomerase contributes to the survival of proliferating plant cells by maintaining telomere length, and that telomere erosion triggers cell death.
Keywords: Telomestatin; Telomerase activity; Cell death; Arabidopsis; Rice;

Most classical phosphotyrosyl phosphatases (PTPs), including the Src homology phosphotyrosyl phosphatase 2 (SHP2) possess a Thr or a Ser residue immediately C-terminal to the invariant Arg in the active site consensus motif (H/V-C-X5-R-S/T), also known as the “signature motif”. SHP2 has a Thr (Thr466) at this position, but its importance in catalysis has not been investigated. By employing site-directed mutagenesis, phosphatase assays and substrate-trapping studies, we demonstrate that Thr466 is critical for the catalytic activity of SHP2. Its mutation to Ala abolishes phosphatase activity, but provides a new substrate-trapping mutant. We further show that the nucleophilic Cys459 is not involved in substrate trapping by Thr466Ala-SHP2 (T/A-SHP2). Mutation of Thr466 does not cause significant structural changes in the active site as revealed by the trapping of the epidermal growth factor receptor (EGFR), the physiological substrate of SHP2, and by orthovanadate competition experiments. Based on these results and previous other works, we propose that the role of Thr466 in the catalytic process of SHP2 could be stabilizing the sulfhydryl group of Cys459 in its reduced state, a state that enables nucleophilic attack on the phosphate moiety of the substrate. The T/A-SHP2 harbors a single mutation and specifically interacts with the EGFR. Since the nucleophilic Cys459 and the proton donor Asp425 are intact in the T/A-SAHP2, it offers an excellent starting material for solving the structure of SHP2 in complex with its physiological substrate.
Keywords: SHP2; Phosphatase; EGFR; Substrate trapping;

Previous studies from this laboratory showed that the action of Osteogenic Protein-1 (OP-1, BMP-7) on osteoblastic cell differentiation could be enhanced by other protein factors, such as Insulin-like Growth Factor (IGF)-I. In the present study, we examined the effects of co-transfection with a combination of the OP-1 and the IGF-I gene on osteoblastic cell differentiation. The results first showed that fetal rat calvaria (FRC) cells transfected with the OP-1 gene under the control of the cytomegalovirus (CMV) promoter showed substantial production of the OP-1 protein. Transfected FRC cells also showed a DNA concentration-dependent increase in alkaline phosphatase (AP) activity, an osteoblastic cell differentiation marker. Von Kossa-positive nodules, a hallmark of bone formation in long-term cultures of bone-derived cells, were also observed in the transfected cells after 26 days in culture, whereas none were observed in control cells. Co-transfection of FRC cells with the combination of the OP-1 and the IGF-I gene resulted in a synergistic stimulation of AP activity. The increase was DNA dose-dependent. The current data show that transfection of OP-1 gene into osteoblastic cells stimulates osteoblastic cell differentiation in vitro. The study further demonstrates the feasibility of employing gene transfer of a second gene in combination with an OP-1 vector to synergistically enhance OP-1 activity.
Keywords: Bone morphogenetic protein; Osteogenic protein-1; IGF-I; Synergy; Cell differentiation; Osteoblast; Co-expression;

Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior by Romina Giacometti; Guadalupe Souto; Susana Silberstein; Luc Giasson; María Leonor Cantore; Susana Passeron (64-72).
We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 °C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.
Keywords: Candida albicans; Protein expression; BCY1 mutant; Subcellular localization;

Synaptotagmin 8 is expressed both as a calcium-insensitive soluble and membrane protein in neurons, neuroendocrine and endocrine cells by Carole Monterrat; Frédéric Boal; Florence Grise; Agnès Hémar; Jochen Lang (73-81).
Synaptotagmins (syt) form a large family of transmembrane proteins and some of its isoforms are known to regulate calcium-induced membrane fusion during vesicular traffic. In view of the reported implication of the isoform syt8 in exocytosis we investigated the expression, localisation and calcium-sensitivity of syt8 in secretory cells. An immunopurified antipeptide antibody was generated which is directed against a C-terminal sequence and devoid of crossreactivity towards syt1 to 12. Subcellular fractionation and immunocytochemistry revealed two forms of synaptotagmin 8 (50 and 40 kDa). Whereas the 40-kDa was present in the cytosol in brain, in PC12 and in clonal β-cells, the 50-kDa form was localised in very typical clusters and partially colocalised with the SNARE protein Vti1a. Moreover, in primary hippocampal neurons syt8 was only found within the soma. Amplification of syt8 by RT-PCR indicated that the observed protein variants were not generated by alternative splicing of the 6th exon and are most likely linked to variations in the N-terminal region. In contrast to the established calcium sensor syt2, endogenous cytosolic syt8 and transiently expressed syt8-C2AB-eGFP did not translocate upon a raise in cytosolic calcium in living cells. Syt8 is therefore not a calcium sensor in exocytotic membrane fusion in endocrine cells.
Keywords: Calcium-sensor; Subcellular distribution; Exocytosis; Translocation; Pancreatic β-cells; Synaptotagmin;

The UDP-galactose (UDP-Gal) transporter present in the Golgi apparatus is a member of a transporter family comprising hydrophobic proteins with multiple transmembrane domains. Co-immunoprecipitation experiments showed that the full-length UDP-Gal transporter protein forms oligomeric structures in the MDCK cell. A ricin-resistant mutant of the MDCK cell line (MDCK-RCAr) is deficient in galactose linked to macromolecules because of a lower UDP-Gal transport rate into the Golgi apparatus. We cloned this mutated protein and found that it contains a stop codon close to the 5′ terminus of its open reading frame. We also detected a shorter splicing variant of the UDP-Gal transporter which contains a 183-nt in-frame deletion in both the wild-type and the mutant mRNA. We showed that the protein, when overexpressed, is localized in the Golgi apparatus and could partially correct the phenotype of the MDCK-RCAr and CHO-Lec8 mutant cell lines. The level of mRNA of the UDP-Gal transporter is much lower (25–30 copies per cell) than those of the CMP-sialic acid transporter (100 copies per cell), UDP-N-acetylglucosamine transporter (80 copies per cell), and GDP-fucose transporter (65 copies per cell). The transcript level of the shorter splicing variant of the UDP-Gal transporter is extremely rare in wild-type MDCK cells (a few copies per cell), but it is significantly increased in the mutant, RCA-resistant cells.
Keywords: UDP-galactose transporter; MDCK; Golgi apparatus; Membrane protein;

Molecular alterations of cells resistant to platinum drugs: Role of PKCα by Sabina C. Righetti; Paola Perego; Nives Carenini; Elisabetta Corna; Laura Dal Bo; Sabrina Cedrola; Caterina A.M. La Porta; Franco Zunino (93-100).
Development of resistance to platinum compounds may involve not only overexpression of defence mechanisms but also alterations in cellular response to the drug-induced genotoxic stress. To investigate the cellular bases of response to platinum compounds, we examined the profile of gene expression of ovarian carcinoma cells exhibiting sensitivity (A2780) or resistance (A2780/BBR3464) to platinum compounds. Using display PCR, we found that acquisition of resistance to the multinuclear platinum complex BBR3464 was associated with modulation of several transcripts, including up-regulation of the major substrate of protein kinase C (PKC), the myristoylated alanine-rich C kinase substrate (MARCKS). This feature was associated with PKCα down-regulation. To explore the role of PKCα in cellular sensitivity to platinum compounds, resistant cells were transfected with a PKCα-containing vector. PKCα-overexpressing resistant cells exhibited a decrease in sensitivity to cisplatin, whereas no significant change in sensitivity to BBR3464 was observed. A number of approaches designed to modulate the function or expression of PKCα support that the isoenzyme may play a role in determining resistance only to cisplatin but not to BBR3464, which is known to activate a different pathway of cell response. In conclusion, in spite of PKCα down-regulation in our model, its regulatory function was not apparently implicated in the development of resistance to platinum compounds and the present results do not support a general role of PKCα as a determinant of the resistance status.
Keywords: Cisplatin; Platinum drug; Resistance; Protein kinase C;

Functional partitioning of epithelial protein kinase CaMKII in signal transduction by Michael Fährmann; Marc-André Kaufhold (101-109).
The examination of the physiological role of CaMKII has made substantial progress in non-epithelial systems but little is known about its function in secretory epithelial cells. A prototypic exocrine cell, the acid secreting gastric parietal cell, largely redistributes its cytoplasmic tubulocisternal membranes (TC) to the secretory apical membrane (SA) after stimulation. We here present a dependence of physiological response on partitioned initial CaMKII activities redistributed between TC and SA. Initial acid secretion after cholinergic stimulation has doubled if activities of phosphatases PP1/PP2A and protein kinase PKC-α were inhibited by the inhibitors calyculin A and Gö 6976. CaMKII appears to be integrated in multienzyme complexes each specific for TC and SA. Therein, PP1/PP2A and PKC-α were found to permanently counteract initial CaMKII activities in different modes of transregulation. Remarkably, a dys-transregulated, hyperactive CaMKII at TC did not result in an increased acid secretion to the same extent. A simple mathematical model to estimate the share of TC- and SA-associated CaMKII activities in cholinergically induced acid secretion suggests that TC-associated CaMKII appears to autoregulate its contribution to the physiological response by a negative feedback control. Subcellular distribution and stimulus-dependent redistribution of domain-associated CaMKII signalosomes indicate a fine balanced, adaptive system to guarantee a stable physiological response.
Keywords: Ca2+/calmodulin-dependent protein kinase; Phosphoprotein phosphatase; Protein kinase C; Exocrine secretion; Signal transduction; Translocation;

The induction of NOS2 expression by the hybrid cecropin A–melittin antibiotic peptide CA(1–8)M(1–18) in the monocytic line RAW 264.7 is triggered by a temporary and reversible plasma membrane permeation by Cristina Arias; Miriam Guizy; Juan R. Luque-Ortega; Esther Guerrero; Beatriz G. de la Torre; David Andreu; Luis Rivas; Carmen Valenzuela (110-119).
There is an increasing awareness of immune cell modulation by antimicrobial peptides. While this process often requires specific receptors for the peptides involved, several reports point out to a receptor-independent process. The cecropin A–melittin hybrid peptide CA(1–8)M(1–18) (KWKLFKKIGIGAVLKVLTTGLPALIS-amide) modifies gene expression in the macrophage line RAW 264.7 in the absence of any previous macrophage priming, suggesting a membrane permeation process. To further analyze the initial steps of this mechanism, we have studied the interaction of the peptide with these cells. Below 2 μM, CA(1–8)M(1–18) causes a concentration-dependent membrane depolarization partially reversible with time. At 2 μM, the accumulation of the SYTOX green vital dye is one half of that achieved with 0.05% Triton X-100. The binding level, as assessed by fluorescein-labeled CA(1–8)M(1–18), varies from 7.7 ± 1.2 to 37.4 ± 3.9 × 106 molecules/cell over a 0.5–4.0 μM concentration range. Electrophysiological experiments with 0.5 μM CA(1–8)M(1–18), a concentration that triggers maximal NOS2 expression and minimal toxicity, show a reversible current induction in the RAW 264.7 plasma membrane that is maintained as far as peptide is present. This activation of the macrophage involves the production of nitric oxide, a metabolite lethal for many pathogens that results from unspecific membrane permeation by antimicrobial peptides, and represents a new mode of action that may open new therapeutic possibilities for these compounds against intracellular pathogens.
Keywords: Antimicrobial peptide; Macrophage; Cecropin A–melittin; Electrophysiology;

Cell internalization and traffic pathway of Clostridium botulinum type C neurotoxin in HT-29 cells by Nobuo Uotsu; Atsushi Nishikawa; Toshihiro Watanabe; Tohru Ohyama; Takashi Tonozuka; Yoshiyuki Sakano; Keiji Oguma (120-128).
The bacterium Clostridium botulinum type C produces a progenitor toxin (C16S toxin) that binds to O-linked sugar chains terminating with sialic acid on the surface of HT-29 cells prior to internalization [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, Biochem. Biophys. Res. Commun. 319 (2004) 327–333] [21]. Based on this, it was hypothesized that the C16S toxin is internalized via clathrin-coated pits. To examine this possibility, the internalized toxin was observed with a fluorescent antibody using confocal laser-scanning microscopy. The confocal images clearly indicated that the C16S toxin was internalized mainly via clathrin-coated pits and localized in early endosomes. The toxin was colocalized with caveolin-1 which is one of the components of caveolae, however, implying the toxin was also internalized via caveolae. The confocal images also showed that the neurotoxin transported to the endosome was transferred to the Golgi apparatus. However, the non-toxic components were not merged with the Golgi marker protein, TGN38, implying the neurotoxin was dissociated from progenitor toxin in endosomes. These results suggested that the C16S toxin was separated to the neurotoxin and other proteins in endosome and the neurotoxin was further transferred to the Golgi apparatus which is the center for protein sorting.
Keywords: Botulinum toxin; Endocytosis; Mucin; Clathrin; Caveolae; HT-29 cell;

In human phagocytic cells, reactive oxygen species (ROS) generation in response to N-formyl-l-Methionyl-l-Leucyl-l-Phenylalanine (fMLF) is largely dependent on cytosolic free calcium concentration ([Ca2+]i). Cyclic ADP-ribose (cADPr) is able to regulate Ca2+ release from intracellular stores through the ryanodine receptor but its potential role in biological responses has so far not been determined. In this study, we examined whether extracellular and intracellular cADPr is required in fMLF-induced [Ca2+]i rise and consequently in the oxidative response in human neutrophil-like HL-60 cells differentiated with dimethylsulfoxide or all-trans-retinoic acid (ATRA). We establish that extracellular cADPr cannot elicit [Ca2+]i elevation. Furthermore, we demonstrate that 8-Br-cADPr, a functional antagonist of cADPr, inhibits Ca2+ entry into HL-60 cells differentiated with ATRA and stimulated with fMLF (95 ± 4 and 148 ± 5 nM respectively, n  = 3). Finally, we show that this partial inhibition of Ca2+ mobilization is unrelated to ROS production (10.0 ± 0.3 vs. 9.6 ± 0.5 A.U., n  = 3). In conclusion, we showed that cADPr can control fMLF-induced Ca2+ influx but is unable to regulate a Ca2+-dependent biological response, i.e. H2O2 production.
Keywords: Ca2+ mobilization; cADPr; fMLF; H2O2 production; HL-60 cells;