Arsenic is widely distributed in nature and all organisms possess regulatory mechanisms to evade toxicity and acquire tolerance. Yet, little is known about arsenic sensing and signaling mechanisms or about their impact on tolerance and detoxification systems. Here, we describe a novel role of the S. cerevisiae mitogen-activated protein kinase Hog1p in protecting cells during exposure to arsenite and the related metalloid antimonite. Cells impaired in Hog1p function are metalloid hypersensitive, whereas cells with elevated Hog1p activity display improved tolerance. Hog1p is phosphorylated in response to arsenite and this phosphorylation requires Ssk1p and Pbs2p. Arsenite-activated Hog1p remains primarily cytoplasmic and does not mediate a major transcriptional response. Instead, hog1⌬ sensitivity is accompanied by elevated cellular arsenic levels and we demonstrate that increased arsenite influx is dependent on the aquaglyceroporin Fps1p. Fps1p is phosphorylated on threonine 231 in vivo and this phosphorylation critically affects Fps1p activity. Moreover, Hog1p is shown to affect Fps1p phosphorylation. Our data are the first to demonstrate Hog1p activation by metalloids and provides a mechanism by which this kinase contributes to tolerance acquisition. Understanding how arsenite/antimonite uptake and toxicity is modulated may prove of value for their use in medical therapy.

Heme {Fe(II)- or Fe(III)-protoporphyrin IX complex [heme(Fe(2+)) or heme(Fe(3+)), respectively]} binds selectively to the 3'-terminal G-quartet of a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The binding affinities of some chemically modified hemes(Fe(3+)) for DNA and the structures of complexes between the modified hemes(Fe(2+)) and DNA, with carbon monoxide (CO) coordinated to the heme Fe atom on the side of the heme opposite the G6 G-quartet, have been characterized to elucidate the interaction between the heme and G-quartet in the complexes through analysis of the effects of the heme modification on the structural properties of the complex. The study revealed that the binding affinities and structures of the complexes were barely affected by the heme modification performed in the study. Such plasticity in the binding of heme to the G-quartet is useful for the versatile design of the complex through heme chemical modification and DNA sequence alteration. Furthermore, exchangeable proton signals exhibiting two-proton intensity were observed at approximately -3.5 ppm in the (1)H nuclear magnetic resonance (NMR) spectra of the CO adducts of the complexes. Through analysis of the NMR results, together with theoretical consideration, we concluded that the heme(Fe(2+)) axial ligand trans to CO in the complex is a water molecule (H2O). Identification of the Fe-bound H2O accommodated between the heme and G-quartet planes in the complex provides new insights into the structure-function relationship of the complex.

Cancer cells produce unique heterogeneous vesicles 1 capable of transferring oncogenic material 2,3 to other cells, 4,5 with the potential of modulating a tumor-supportive environment. [6][7][8] We have previously reported the presence of lipid-enriched, membrane-bound subcellular vesicles at the periphery of acute lymphoblastic leukemia (ALL) cell lines. 9,10 We now extend these findings to describe heterogeneous anucleate vesicles released into extracellular fluids in vitro and in vivo by primary B-cell precursor (BCP) ALL blasts and cell lines. Leukemic extracellular vesicles (LEVs) were internalized by stromal cells, and induced a metabolic switch.Extracellular vesicles (EVs) are enclosed in lipid bilayers originating from the cell of origin, released by both normal and cancer cells.1 Here, the BCP cell-specific membrane protein CD19 present within membrane lipid rafts 11 was used to identify the cell of origin of EVs in clinical samples. We directly compared plasma samples from CD19 1 primary BCP-ALL bone marrow aspirates at diagnosis containing .95% malignant blasts with matched remission samples obtained after 28 days of therapy ( Figure 1A LEVs ( Figure 1E). The effect of LEV internalization by BMSCs was investigated in the human mesenchymal stem cell line HS5 14 exposed to LEVs released by the BCP-ALL cell lines SD1 and NALM6. Proliferation and viability assays revealed no significant differences from control ( Figure 2A). Despite a sustained increase in AKT phosphorylation over 24 hours ( Figure 2B), nonsignificant reductions in adenosine triphosphate (ATP) concentrations were observed ( Figure 2C). Next, the 2 major energyproducing pathways of the cell and parameters of metabolism were assessed. At 24 hours, HS5 1 LEVs showed a reduced oxygen consumption rate (OCR) compared with control, were less sensitive to the inhibition of ATP by oligomycin, and did not change OCR when electron transport from ATP generation in the mitochondria was uncoupled ( Figure 2D). Disrupting the electron transport chain (rotenone/antimycin A) reduced OCR to a comparable level in all cells, suggesting that the rate of oxygen consumption from nonmitochondrial sources was comparable. HS5 1 LEV have a significantly reduced spare respiratory capacity, an indicator of a decreased ability to respond to stress or metabolic challenge ( Figure 2D). Overall, these results suggest that uptake of LEVs significantly reduced mitochondrial respiration in recipient stromal cells.In the absence of glucose, HS5 and HS5 1 LEVs had comparable extracellular acidification rates (ECARs) ( Figure 2E). In the presence of glucose, HS5 1 LEVs initiated a sharp increase in ECAR compared with control (;fivefold), suggesting a higher glycolytic rate. Inhibiting ATP synthase increased ECAR in both HS5 1 LEVs and controls, but more sharply in the latter. Following the addition of 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, ECARs returned to base levels in both control and LEV-exposed cells. Thus, in the presence of glucose, LEV-exposed HS5 s...

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Toxic metals are ubiquitous in the environment and all organisms possess systems to evade toxicity and acquire tolerance. The Saccharomyces cerevisiae AP-1-like protein Yap8p (systematic name YPR199c; also known as Acr1p and Arr1p) confers arsenic tolerance by stimulating enhanced transcription of the arsenic-specific detoxification genes ACR2 and ACR3. Here, we report that Yap8p is regulated at the level of degradation. We show that Yap8p is stabilized in arsenite-exposed cells in a time- and dose-dependent manner. Yap8p degradation proceeds through the ubiquitin-proteasome pathway and is dependent on the ubiquitin-conjugating enzyme Ubc4p. Further, we show that mutants that are defective in the ubiquitin-proteasome pathway display increased Yap8p levels and elevated expression of the Yap8p gene-target ACR3. Yap8p forms homodimers in vivo but dimerization is not regulated by arsenite. Instead, arsenite-stimulated Yap8p stabilization and transcriptional activation of ACR3 requires critical cysteine residues within Yap8p. Collectively, our data is consistent with a model where Yap8p is degraded by the ubiquitin-proteasome pathway in untreated cells, whereas arsenite-exposure results in Yap8p stabilization and gene activation. In this way, regulated degradation contributes to Yap8p control.