NSNM exposure leads to degradation of Sml1 without activating RNR genes or Rad52 foci formation.

<p>(A) Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot analysis with indicated antibodies. Blotting with antibodies against Tbp and Rap1 or Ponceau S staining of representative blot were used as loading controls. (B) Sml1-YFP tagged strain were treated with NSNM (100 μM) for 3 h. For control same strains were treated with MMS (0.03%), images were taken as described in materials and methods. (C and D) Growth Assay; wild type and different mutant yeast strains were spotted onto control SCA (DMSO) plates or SCA plates containing 100, 200 or 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. (E) NSNM exposure does not lead to Rad52 foci formation. Rad52-YFP tagged yeast strain was treated with 100 μM NSNM for 3 h, same strain was treated with 0.03% MMS (control) prior to visualization of foci by confocal microscopy.</p

Screening of yeast deletion-mutants for NSNM sensitivity.

<p>A–E) Growth Assay; yeast deletion mutants of various pathways were grown up to log phase. 3 μl of each undiluted and 10-fold serially diluted culture was spotted onto control SCA plates and SCA plates containing 100, 200 and 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. Mutant yeast strains of A) Histone tails, B) HATs and HDACs, C) Molecular chaperones, D) Protein-ubiquitination pathways, E) TOR pathway.</p

NSNM inhibits growth of wild type yeast cells in a dose-dependent manner.

<p>A) Growth curves of wild-type <i>S. cerevisiae</i> in the presence or absence of drug. Exponentially growing yeast cultures of the wild-type strain were treated with the indicated concentrations of NSNM. Growth was monitored by measuring OD₆₀₀ at regular intervals for 8 h. B) Growth Assay; 1588-4C (Wild-type) was grown up to log phase. 3 μl of each undiluted and 10-fold serially diluted culture was spotted onto control SCA plates and SCA plates containing 100, 200 or 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. C) Clonogenic assay; equal number of cells from mid-log phase of untreated and NSNM treated cultures (3 h) were spread on standard SCA plates in triplicate. All plates were incubated at 30°C and the colony forming ability was analyzed after 36 h. Number of colonies were counted and shown in the form of bar diagram. D and E) FACS analysis, showing the effect of the NSNM on yeast cell cycle. Wild-type cells were cultured in SC medium to exponential phase and treated with alpha factor to synchronize all cells in G1 phase. After synchronization cells were released in either DMSO (control) or 50 μM NSNM containing media. The culture was sampled at indicated time points and cellular DNA content was analysed by FACS.</p

NSNM causes depletion in total glutathione levels and increase intracellular ROS levels.

<p>(A) Wild type cells were grown in DMSO or indicated concentration of NSNM for 3 h. GSH, GSSG, and the GSH: GSSG ratios were determined. Values are means S.D. of three independent cultures. (B & C) Mitochondrial membrane permebility and reactive oxygen species (ROS) production detected by MitoTracker and DCF-DA respectively in control cells and cells treated with 50 or 100 μM of NSNM for 3 h. Cells treated with 5 mM H₂O₂ served as positive control. Upper panel of (C) shows phase contrast microscopic images; the lower panel show florescence microscopic image of the same cells. (D) Wild-type yeast strain grown in SC media and treated with increasing concentration of NSNM (25, 50 or 100 μM) for 1 3 h. Yeast cells were processed for FACS analysis after staining with either DCF-DA or MitoTracker Red. % value depicts the proportion of cells showing fluorescence after staining with indicated dyes.</p

NSNM induces alterations in histone modifications and makes adduct with histone H3 and H4.

<p>A) Wild-type (1588-4C) cells were cultured up to log phase and treated with increasing concentrations of NSNM (0, 10, 25, 50, 100 μM) for 3 hr. Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot analysis using indicated antibodies. B) Wild type yeast cells were treated with DMSO and 100 μM NSNM for 3 h at OD₆₀₀ (0.8) and equal numbers of cells were processed for MNase digestion. MNase was used at different concentrations (0, 50, 100, 200, 300, 500 and 1000 U/ml). Samples were run on 1.2% agarose gel, together with DNA ladder, and stained with ethidium-bromide. C) Core histones were incubated with increasing concentrations of NSNM for 1 h at 37 °C. Proteins were resolved by 12% SDS-PAGE. The interaction of NSNM with histones was analyzed by probing with histone H3 and H4 antibodies. Arrow indicates the appearance of high molecular weight band detected by H3 and H4 antibody respectively. * represents non-specific band detected by H3 antibody.</p

SODs deletions are hypersensitive to the NSNM while GSH supplementation rescues the effect.

<p>(A) Growth Assay; Wild type, <i>sod1Δ</i> or <i>sod2Δ</i> was grown up to log phase. 3 μl of each undiluted and 10-fold serially diluted culture was spotted onto control SCA plates, SCA plates containing 100, 200, 300 μM NSNM, and SCA plates impregnated with GSH (10 mM) in combination with 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. (B &C) Wild-type yeast strain grown in SC media supplemented with or without 10 mM GSH for 1 h followed by exposure to 100 μM NSNM for 3 h. Yeast cells were processed for FACS analysis after staining with either DCF-DA (B) or MitoTracker Red (C). % value depicts the proportion of cells showing florescence after staining with indicated dyes.</p

Depletion of Cellular Iron by Curcumin Leads to Alteration in Histone Acetylation and Degradation of Sml1p in <em>Saccharomyces cerevisiae</em>

<div><p>Curcumin, a naturally occurring polyphenolic compound, is known to possess diverse pharmacological properties. There is a scarcity of literature documenting the exact mechanism by which curcumin modulates its biological effects. In the present study, we have used yeast as a model organism to dissect the mechanism underlying the action of curcumin. We found that the yeast mutants of histone proteins and chromatin modifying enzymes were sensitive to curcumin and further supplementation of iron resulted in reversal of the changes induced by curcumin. Additionally, treatment of curcumin caused the iron starvation induced expression of <i>FET3, FRE1</i> genes. We also demonstrated that curcumin induces degradation of Sml1p, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. The degradation of Sml1p was mediated through proteasome and vacuole dependent protein degradation pathways. Furthermore, curcumin exerts biological effect by altering global proteome profile without affecting chromatin architecture. These findings suggest that the medicinal properties of curcumin are largely contributed by its cumulative effect of iron starvation and epigenetic modifications.</p> </div

Curcumin inhibits growth of yeast cells and its effect is antagonized by iron supplementation.

<p>A) Growth Assay; 1588-4C (Wild-type) was grown up to log phase. 3 µl of each undiluted and 10-fold serially diluted culture was spotted onto control SCA plates, SCA plates containing 200, 400, 600 μM curcumin, and SCA plates impregnated with iron (100 μM) in combination with 600 μM curcumin. All plates were incubated at 30°C for 72 hr and photographed. B) Viability Assay; 1588-4C (Wild-type) was cultured up to mid-log phase and treated with 10, 25, 50, 100 and 200 μM curcumin for 3 hr and cells were stained with 0.3% methylene blue for checking viability. Untreated and heat killed cells were taken as negative and positive controls respectively; viability was observed under light microscope (400X) and photographed. C) Clonogenic assay; equal number of cells from mid-log phase of untreated and methylene blue stained curcumin (400 μM) treated cultures were spread on standard SCA plates in triplicate. All plates were incubated at 30°C and the colony forming ability was analyzed after 36 hr. No. of colonies were counted and shown in the form of bar diagram. D) Viability Assay; 1588-4C (Wild-type) was cultured up to mid-log phase, treated with 200 μM curcumin or iron (100 μM) in combination with 200 μM curcumin. A small number of cells were collected and stained with 0.3% methylene blue at different time points (10, 30, 60, 90 and 120 min) of curcumin treatment for viability examination. Untreated and heat killed cells were taken as negative and positive controls respectively; viability was observed under light microscope (400X) and photographed.</p

Proposed model for action of curcumin in budding yeast

<p>. Curcumin causes iron starvation by chelating it which leads to transcriptional alteration of iron-regulated genes. Histone hypo-acetylation by curcumin results in delayed growth phenotype. Curcumin induces Sml1p degradation through vacuole and proteasome-mediated protein degradation pathways. We propose that after Sml1p degradation the released Rnr1 associates with Rnr2 and Rnr4 to form an active RNR enzyme allowing the production of dNTPs.</p

Curcumin alters global proteomics.

<p>Whole cell lysate was prepared by disrupting mid-log phase untreated and curcumin (400 μM) treated cultures using chilled glass beads. Protein extracts were prepared by precipitation and clean-up as mentioned in ‘Materials and methods’. A) Equal quantity of protein extracts were resolved by SDS-PAGE. B) 200 µg of total cellular proteins from untreated and curcumin-treated cultures was resolved by two-dimensional gel electrophoresis; first by isoelectric focusing (IEF) on a 7 cm long Immobiline^TM pH 3–10 DryStrip, followed by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R-250. C) Enlarged images of the upper left (Inset I), upper right (Inset II), lower left (Inset III), and lower right (Inset IV) regions of the two-dimensional gels containing proteins from untreated and curcumin-treated cells are shown. The differentially expressed spots are indicated by red circles.</p