Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking

RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast

Folding-competent and folding-defective forms of Ricin A chain have different fates following retrotranslocation from the endoplasmic reticulum

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTAΔ), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTAΔ, although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate

Intracellular retention of ABL kinase inhibitors determines commitment to apoptosis in CML cells

Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by highdose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs.Daniel B. Lipka, Marie-Christine Wagner, Marek Dziadosz, Tina Schnöder, Florian Heidel, Mirle Schemionek, Junia V. Melo, Thomas Kindler, Carsten Müller-Tidow, Steffen Koschmieder and Thomas Fische

Intracellular trafficking and in vivo toxicity of Ricin A chain (RTA) in yeast

Das Pflanzentoxin Ricin aus Ricinus communis gehört zur Gruppe der A/B-Toxine, die aus einer katalytischen A-Kette und einer oder mehreren B-Ketten aufgebaut sind. Das Toxin gelangt über die Bindung der B-Kette an Galaktose-haltige Strukturen auf der Zelloberfläche endozytotisch in die Zielzelle und wird in einem retrograden Prozess bis zum Endoplasmatischen Retikulum (ER) transportiert. Bislang wird angenommen, dass der eigentliche ER/Cytosol-Export von RTA unter Ausnutzung der ER-assoziierten Degradation (ERAD) erfolgt. Im Cytosol fungiert die katalytische A-Kette als RNA N-Glykosidase und depuriniert ribosomale RNA, was eine Hemmung der Proteinbiosynthese und letztendlich die Apoptose der Zelle bewirkt. Anhand der intrazellulären Expression verschiedener cytotoxischer RTA-Derivate im ER-Lumen von Hefezellen wurde der molekulare Retrotranslokations-Mechanismus der Ricin A Kette näher untersucht. Durch die Analyse ausgewählter Hefe-Deletionsmutanten konnten zelluläre Komponenten identifiziert werden, die in diesem Prozess sowie für die in vivo Toxizität von RTA eine entscheidende Rolle spielen. Die Expression der beiden ER-luminalen RTA-Varianten K1- bzw. K28-RTA induzierte ein vermindertes Wachstum der transformierten Wildtyp-Hefen, während die in vivo Expression der mutierten RTA-Derivate K1- bzw. K28-RTAE177D einen resistenten Phänotyp verursachte. Die in vivo Expression der Konstrukte K1-/K28-RTA konnte mittels radioaktiven "Labeling"-Experimenten gezeigt werden, im Gegensatz zu der ER-luminalen Toxin-Variante Kar2-RTA konnte jedoch der Nachweis einer posttranslationalen RTA-Glykosylierung im ER nicht erbracht werden, was einen Hinweis auf einen zumindest deutlich eingeschränkten Import von Ricin A in das ER lieferte. Für beide Konstrukte konnte ein Eintritt in das Endoplasmatische Retikulum indirekt durch die verminderte in vivo Toxizität in Mutanten mit Defekten im posttranslationalen ER-Import, im Signalpeptidase-Komplex sowie in cytosolischen Chaperon-Mutanten bewiesen werden. Das Protein L12 der ribosomalen GTPase-Domäne spielte eine essentielle Rolle für die in vivo Toxizität von Ricin A. Ein Transport des Toxins über den Golgi-Apparat schien für dessen in vivo Toxizität nicht ausschlaggebend zu sein. Im Gegensatz zur ER-luminalen Variante Kar2-RTA nutzen die Konstrukte K1-/K28-RTA sowohl Komponenten des Hrd1-Komplexes (ERAD-L), als auch des Doa10-Komplexes (ERAD-C). Die Abwesenheit klassischer ERAD-Komponenten wie Hrd1p, Hrd3p, Der1p, Usa1p, Ubx2p, Ubc7p, Cue1p, Ubc1p, Doa10p und/oder Cne1p führte zu einem drastischen Aktivitätsverlust von RTA. Eine Beteiligung des Cdc48-Komplexes konnte ebenfalls durch indirekte Hinweise bestätigt werden. Die Polyubiquitinierung scheint eine entscheidende Funktion für die in vivo Toxizität und die Retrotranslokation von RTA einzunehmen. Wurde die Polyubiquitinierung der Konstrukte K1-/K28-RTA durch Austausch der internen Lysinreste oder durch Überexpression einer mutierten Ubiquitin-Form, die keine Polyubiquitin-Ketten mehr ausbilden kann, verhindert, so kam es zu einer deutlich verminderten Toxizität. Eine in vivo Interaktion von RTA mit Ubiquitin konnte mittels bimolekularer Fluoreszenzkomplementation (BiFC) nachgewiesen werden. Es konnte zudem eine "Screening"-Methode etabliert werden, mit der es möglich ist, eine Vielzahl von Hefe-"Knock-out"-Mutanten effektiv und in relativ kurzer Zeit auf Sensitivität gegen RTA zu untersuchen, und somit weitere Komponenten zu identifizieren, die am intrazellulären Transport und der in vivo Toxizität von RTA beteiligt sind.The plant toxin ricin from Ricinus communis is a family member of so-called A/B toxin in which a catalytic A subunit is associated with one or more cell-binding B subunits. After binding of the B subunit to galactose-containing cell surface receptors, the holotoxin is taken up by endocytosis and delivered to the endoplasmic reticulum via retrograde transport through the Golgi. For ER-to-cytosol dislocation the toxin probably exploits components of the ER-associated degradation machinery (ERAD). Within the cytosol the A subunit acts as RNA N-glykosidase that depurinates ribosomal RNA inducing inhibition of protein synthesis and apoptosis. In this study, intracellular expression of different ER luminal RTA variants has been used to characterize toxin retrotranslocation from the ER into the cytosol of intoxicated yeast cells. Selected yeast deletion mutants were screened for toxin sensitivity in order to identify cellular components crucial for the in vivo toxicity of ricin A. In vivo functionality of the different RTA constructs was tested by in vivo toxicity assays. Thus it could be demonstrated that chimeric toxin variants with wildtype RTA are significantly more toxic for yeast cells than variants containing mutated RTAE177D. In vivo expression of each RTAE177D variant was demonstrated by [35S] labelling experiments. In contrast to the ER luminal variant Kar2-RTA, posttranslational glycosylation of K1- and/or K28-RTA was not detectable, indicating failing or inefficient ER import when driven by either K1 or K28 signal sequence. Entry of K1-/K28-RTA into the yeast ER lumen could be verified by a significant decrease in toxicity after in vivo expression in mutants either defective in posttranslational ER import, in signal peptidase complex components and/or in cytosolic chaperones. For both RTA constructs access to the Golgi was not essential for toxin dislocation and/or in vivo toxicity. In contrast to the ER luminal variant Kar2-RTA, the toxin variants K1-/K28-RTA both required components of the Hrd1- and Doa10-complex for retrotranslocation. Lack of classical ERAD components such as Hrd1p, Hrd3p, Der1p, Usa1p, Ubx2p, Ubc7p, Cue1p, Ubc1p, Doa10p and/or Cne1p as well as Cdc48 complex components was accompanied by a complete loss in RTA toxicity. Polyubiquitination of RTA was shown to be essential for both in vivo toxicity and retrotranslocation. Preventing polyubiquitination of RTA either by substitution of all internal lysine residues or by simultaneous overexpression of mutant ubiquitin (Ub-RR48/63), unable to form polyubiquitin chains, resulted in a significant decrease in the in vivo toxicity of ricin A. To identify new components involved in RTA toxicity and/or intracellular transport, a genetic screen based on GFP fluorescence was established which allows comprehensive identification and analysis of S. cerevisiae knock-out mutants defective in ER-to-cytosol retrotranslocation of RTA

Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking

RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast

Memantine potentiates cytarabine-induced cell death of acute leukemia correlating with inhibition of Kv1.3 potassium channels, AKT and ERK1/2 signaling

Abstract Background Treatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated Kv1.3 potassium channels, but their role in acute leukemia is unclear. We reported that Kv1.3 channels of lymphocytes are blocked by memantine, which is known as an antagonist of neuronal N-methyl-D-aspartate type glutamate receptors and clinically applied in therapy of advanced Alzheimer disease. Here we evaluated whether pharmacological targeting of Kv1.3 channels by memantine promotes cell death of acute leukemia cells induced by chemotherapeutic cytarabine. Methods We analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients’ acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of Kv1.3 channels and membrane depolarization by memantine. Cell death was determined with propidium iodide, Annexin V and SYTOX staining and cytochrome C release assay. Molecular effects of memantine co-treatment on activation of Caspases, AKT, ERK1/2, and JNK signaling were analysed by Western blot. Kv1.3 channel expression in Jurkat cells was downregulated by shRNA. Results Our study demonstrates that memantine inhibits Kv1.3 channels of acute leukemia cells and in combination with cytarabine potentiates cell death of acute lymphoid and myeloid leukemia cell lines as well as primary leukemic blasts from acute leukemia patients. At molecular level, memantine co-application fosters concurrent inhibition of AKT, S6 and ERK1/2 and reinforces nuclear down-regulation of MYC, a common target of AKT and ERK1/2 signaling. In addition, it augments mitochondrial dysfunction resulting in enhanced cytochrome C release and activation of Caspase-9 and Caspase-3 leading to amplified apoptosis. Conclusions Our study underlines inhibition of Kv1.3 channels as a therapeutic strategy in acute leukemia and proposes co-treatment with memantine, a licensed and safe drug, as a potential approach to promote cytarabine-based cell death of various subtypes of acute leukemia

Synergistic killing of FLT3ITD-positive AML cells by combined inhibition of tyrosine-kinase activity and N-glycosylation

Fms-like tyrosine kinase 3 (FLT3) with internal tandem duplications (ITD) is a major oncoprotein in acute myeloid leukemia (AML), and confers an unfavorable prognosis. Interference with FLT3ITD signaling is therefore pursued as a promising therapeutic strategy. In this study we show that abrogation of FLT3ITD glycoprotein maturation using low doses of the N-glycosylation inhibitor tunicamycin has anti-proliferative and pro-apoptotic effects on FLT3ITD-expressing human and murine cell lines. This effect is mediated in part by arresting FLT3ITD in an underglycosylated state and thereby attenuating FLT3ITD-driven AKT and ERK signaling. In addition, tunicamycin caused pronounced endoplasmatic reticulum stress and apoptosis through activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of the gene encoding CCAAT-enhancer-binding protein homologous protein (CHOP). PERK inhibition with a small molecule attenuated CHOP induction and partially rescued cells from apoptosis. Combination of tunicamycin with potent FLT3ITD kinase inhibitors caused synergistic cell killing, which was highly selective for cell lines and primary AML cells expressing FLT3ITD. Although tunicamycin is currently not a clinically applicable drug, we propose that mild inhibition of N-glycosylation may have therapeutic potential in combination with FLT3 kinase inhibitors for FLT3ITD-positive AML

PLCγ1 suppression promotes the adaptation of KRAS-mutant lung adenocarcinomas to hypoxia.

Mutant KRAS modulates the metabolic plasticity of cancer cells to confer a growth advantage during hypoxia, but the molecular underpinnings are largely unknown. Using a lipidomic screen, we found that PLCγ1 is suppressed during hypoxia in KRAS-mutant human lung adenocarcinoma cancer cell lines. Suppression of PLCγ1 in hypoxia promotes a less oxidative cancer cell metabolism state, reduces the formation of mitochondrial reactive oxygen species and switches tumour bioenergetics towards glycolysis by impairing Ca2+ entry into the mitochondria. This event prevents lipid peroxidation, antagonizes apoptosis and increases cancer cell proliferation. Accordingly, loss of function of Plcg1 in a mouse model of KrasG12D-driven lung adenocarcinoma increased the expression of glycolytic genes, boosted tumour growth and reduced survival. In patients with KRAS-mutant lung adenocarcinomas, low PLCγ1 expression correlates with increased expression of hypoxia markers and predicts poor patient survival. Thus, our work reveals a mechanism of cancer cell adaptation to hypoxia with potential therapeutic value

Experimental set-up for analysis of induction of apoptosis upon HD-TKI exposure.

<p>(<b>A</b>) Cells were seeded at a density of 5×10⁴ cells/ml in a total volume of 2 ml in RPMI 1640 supplemented with 10% FCS. Cells were treated for 2 h with TKI as indicated. Then, cells were washed twice with 2 ml PBS at room temperature and replated in fresh cell culture media (2 ml final volume). Cells exposed to 0.35% DMSO served as controls. 24 h after start of TKI exposure percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining. (<b>B</b>) To analyze for residual TKI activity upon HD-TKI pulse exposure, a second and third drug wash-out procedure (each consisting of 2×2 ml PBS washing) was performed: Cells were treated with TKI for 2 h. Cells initially pulse-exposed to HD-TKI were washed twice with 2 ml PBS at room temperature and replated in 2 ml fresh media (density: 5×10⁴ cells/ml) as described in (a) (“<i>1x”</i>). To test for residual TKI-activity, the cell culture supernatant was transferred to previously untreated cells <i>(“S1”)</i>, which were subsequently incubated for 24 h. Two hours after replating, a second drug wash-out was performed (2×2 ml PBS). Cells were again replated in 2 ml fresh media (“<i>2x”</i>). Again supernatants were transferred to previously untreated cells (“<i>S2”</i>). This procedure was repeated for a third time (“<i>3x”, “S3”</i>).</p

Repetitive washing prevents apoptosis after high-dose TKI pulse-exposure.

<p>To analyze for residual TKI activity upon HD-TKI pulse-exposure, we employed repetitive wash-out procedures and exposure of previously untreated cells to cell culture supernatants. Therefore, 5×10⁴ cells/ml were seeded in a total volume of 2 ml in RPMI 1640 supplemented with 10% FCS and treated with TKI as indicated for 2 hours. Cells exposed to 0.35% DMSO served as controls (“M”). Then, cells were washed and replated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g001" target="_blank"><b>Figure 1B</b></a> (“1x”, “2x”, “3x”; upper panel). To test for residual TKI-activity, the cell culture supernatants were transferred to previously untreated cells (“S1”, “S2”, “S3”; lower panel). For further details of the experimental procedure please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g001" target="_blank"><b>Figure 1B</b></a>. Percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining 24 hours after start of TKI treatment and is depicted for Ba/F3-BCR-ABL cells (<b>A</b>) and K562 cells (<b>B</b>), respectively. Experiments were performed in triplicate. Data are presented as mean percentage of cells in subG1 phase + SEM.</p