Structural changes in 20S proteasomes after HAMLET treatment <i>in vitro</i>.

<p>(A, B) <i>In vitro</i> fragmentation of human 20S proteasomes after 20 minutes incubation with HAMLET (7 µM), compared to the α-lactalbumin control. (A) The proteins were separated by SDS-PAGE and gels were stained with Amido black. Changes in 20S proteasome subunits are indicated by arrows. The subunits identified by mass spectrometry are shown in the table. (B) Western Blot of samples from <i>in vitro</i> mixing experiment with 20S proteasomes and HAMLET or α-lactalbumin using antibodies against the 20S proteasome core subunits. (C) MALDI-TOF mass spectrometry of intact 20S proteasomes (top panel) and major changes after HAMLET treatment (bottom panel), indicated by arrows.</p

Resistance of HAMLET to degradation by proteasomal enzymes.

<p>(A, B) HAMLET, partially unfolded α-lactalbumin (apo α-lactalbumin) or native α-lactalbumin was exposed to trypsin (E∶S 1∶50, by weight) (A) or chymotrypsin (E∶S 1∶100, by weight) (B) <i>in vitro</i> and fragments were detected by SDS-PAGE. Native α-lactalbumin was in 200 µM calcium acetate whereas partially unfolded α-lactalbumin was in 200 µM EDTA. Arrows indicate time points when all HAMLET was degraded. (C) HAMLET or partially unfolded α-lactalbumin (apo α-lact) (both 7 µM) were mixed with 6 µg of human erythrocyte 20S proteasomes and incubated for 2 hours at 37°C. The proteins were separated by SDS-PAGE and visualized by silver staining. MG132 was used to inhibit proteasome activity. The arrows indicate the molecular sizes of full length apo α-lactalbumin/HAMLET (14 kDa) and a smaller fragment (∼7 kDa).</p

HAMLET inhibits proteasome activity.

<p>(A) Proteasome activity was monitored <i>in vitro</i> as chymotrypsin-dependent suc-LLVY-AMC cleavage (50 µM) over time and MG132 was used as proteasome inhibitor. Except for the first 10 minutes HAMLET reduced proteasome activity compared to the control. (B) Inhibition of proteasome activity in A549 cells. Cytoplasmic extracts were prepared from cells treated with HAMLET or native α-lactalbumin (34 µM) after 1, 3 and 6 hours of incubation and enzymatic activity was quantified by suc-LLVY-AMC cleavage (50 µM). MG132 was used to inhibit proteasome function.</p

Interaction of HAMLET with proteasomes <i>in vitro</i> and in tumor cells.

<p>(A) Binding of HAMLET to 20S proteasome subunits. 20S proteasomes were denatured, separated by SDS-PAGE and stained with Amido black or silver staining or blotted onto PVDF membranes. Left panels: Membranes were overlaid with 0.14 mM HAMLET and binding was detected by immunoblot using anti-α-lactalbumin antibodies. Right panels: Membranes were overlayed with ¹²⁵I-HAMLET and binding was detected by autoradiography. (B) Binding of HAMLET to 20S proteasome β5 subunit on a protein array. Protein arrays were probed with Alexa Fluor 568-labeled HAMLET and fluorescence intensity was measured. HAMLET bound to the 20S proteasome β5 subunit in a concentration-dependent manner. (C) Co-immunoprecipitation of lysates from HAMLET-treated A549 cells (34 µM, 1 hour) with anti-α-lactalbumin antibodies (IP anti-α-lact) or no antibodies (IP mock). Lysates and precipitates were examined by Western Blot using antibodies against the 20S proteasome core subunits. A very weak band was detected at ∼30 kDa. (D) Confocal images showing co-localization of Alexa Fluor 568-labeled HAMLET (34 µM, 1 hour) and 20S proteasomes (antibody against 20S proteasome core subunits) in the cell periphery and cytoplasm of A549 cells.</p

HAMLET is a complex of partially unfolded α-lactalbumin and oleic acid.

<p>(A) HAMLET is formed when native α-lactalbumin releases the strongly bound Ca²⁺ and exposes a fatty-acid binding site, which permits oleic acid (C18:1, 9 <i>cis</i>) to interact and stabilize the partially unfolded state. The figure was generated using the crystal structure of human α-lactalbumin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005229#pone.0005229-Acharya1" target="_blank">[39]</a> and MOLMOL 2K.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005229#pone.0005229-Koradi1" target="_blank">[40]</a>. (B) Near-UV CD spectra were recorded in sodium phosphate buffer without EDTA. Native α-lactalbumin showed the expected minimum at 270 nm arising from the tyrosine and a maximum at 293 nm arising from the tryptophan residues. In HAMLET, α-lactalbumin showed a loss of signal at 270 nm and at 293 nm, typical of partial unfolding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005229#pone.0005229-Svensson1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005229#pone.0005229-Svensson2" target="_blank">[16]</a>. Apo α-lactalbumin was obtained by EDTA treatment and showed a similar loss of signal. (C) Effect of HAMLET on tumor cells and healthy cells. Glioblastoma cells and kidney carcinoma cells treated with HAMLET (34 µM, 1 and 3 hours) died while normal astrocytes and renal epithelial cells (HRTEC) remained viable. (D) Difference in uptake of HAMLET and α-lactalbumin by tumor cells. Lung carcinoma cells (A549) were exposed to 34 µM of Alexa Fluor 568-labeled HAMLET or α-lactalbumin (both red). (E) Quantification by flow cytometry of HAMLET or α-lactalbumin uptake by A549 cells (N = 10,000) after 1 and 3 hours of incubation.</p

Identification of cytoskeleton-associated proteins whose depletion induces non-apoptotic cancer cell death.

<p>(A, B) MCF7 (A), HeLa (B) and U-2-OS (B) cells were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or three independent siRNAs against the indicated targets individually (8 nM; A) or in pools (3×6.67 nM; B). Cell density was measured after 72 h by the MTT reduction assay. (C) MCF7-Bcl-2 or MCF7-pCEP (control) cells were transfected as in (B). <i>Left bottom</i>, After 96 h, cell death was determined by counting Hoechst 33342-stained cells with condensed nuclei (three random fields of 100 cells). TNF (20 ng/ml, 24 h) served as a positive control for Bcl-2 sensitive apoptotic cell death. <i>Left top</i>, Western blot confirming overexpression of Bcl-2 in untreated MCF7-Bcl-2 cells. <i>Right</i>, Examples of images of Hoechst 33342-stained nuclei of MCF7-pCEP and MCF7-Bcl-2 cells 96 h after transfection with indicated siRNAs. (D) MCF7 cells were treated as in (B). <i>Left</i>, After 60 h, DNA was stained with propidium iodide and cell cycle distribution analyzed by flow cytometry (FL-2A). <i>Right</i>, Examples of histograms showing cell cycle distribution of cells 60 h after transfection with indicated siRNAs. Values represent means + SD of three independent experiments (A, C, D) or triplicates in one representative experiment (B, n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells or as indicated (C).</p

Changes in 20S proteasome staining after HAMLET treatment of carcinoma cells.

<p>(A–C) Changes in the staining of 20S proteasome subunits in A549 cells after HAMLET treatment (34 and 45 µM, 1 hour). Cells were stained with antibodies against the 20S proteasome core subunits (α5, α7, β1, β5, β5i and β7), antibodies against the β1 subunit or antibodies against the β5 subunit. (A) Confocal images showing increased core and β1 subunit staining, and decreased β5 subunit staining after HAMLET treatment (45 µM, 1 hour). (B) Fluorescence intensity was quantified in control and HAMLET-treated cells (34 and 45 µM, 1 hour). (C) The increase in core subunit staining after HAMLET was blocked by proteasome inhibition with MG132. (D) Western blot of cell extracts from cells treated with HAMLET (34 µM) for 1 and 6 hours, showing loss of 20S proteasomes staining after 6 hours. Antibodies against the 20S proteasome core subunits were used.</p

Effect of the identified siRNAs on autophagy and dextran uptake.

<p>(A–D) tfLC3-MCF7 cells (A, B) or MCF7 cells (C, D) were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or indicated siRNA pools (3×6.67 nM). (A, B) After 48 h, tfLC3-MCF7 cells were analyzed by confocal microscopy. Representative images (A; <i>Bars</i>, 10 µm) and quantification of puncta (B) are shown. Raptor siRNA (RPTOR) served as a control for increased autophagic flux. Closed arrows indicate AVd, open arrows indicate AVi. (C) After 60 h, the level of p62/SQSTM1 (p62), which is degraded by autophagy, was examined by Western blot. Rapamycin (20 nM, 4 h) was used to induce autophagy. Numbers represent p62 levels as percentage of the level in untreated control siRNA-transfected cells. (D) <i>Top</i>, After 60 h, MCF7 cells were treated with 100 µg/ml Alexa Fluor 488-dextran (dextran-488) for 1 h and analyzed by flow cytometry (FL1-H). The threshold for high intensity staining was defined so that 88% of control siRNA-transfected cells were below. <i>Bottom</i>, Example histograms showing dextran-488 content of cells transfected with control or KIF20A siRNA. M1 = gate for high intensity staining. Values represent means + SEM of 20 cells in one representative experiment (B, n = 3) or means + SD of three independent experiments (D). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells.</p

Identification of Cytoskeleton-Associated Proteins Essential for Lysosomal Stability and Survival of Human Cancer Cells

<div><p>Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, <em>i.e.</em> increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, <em>i.e.</em> photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.</p> </div

Summary of cellular changes induced by the depletion of survival-associated motor proteins.

1<p>CC, cell cycle.</p>2<p>VAC, volume of the acidic compartment (late endosomes and lysosomes).</p>3<p>photo-oxidation-induced lysosomal leakage.</p>4<p>−, decrease, <i>P</i><0.05; +, increase, <i>P</i><0.05; ±, no change; () 0.05<<i>P</i><0.10.</p>5<p>It should be noted that these data might be affected by cell death that starts already 50 hours after the transfection with KIF21A siRNAs.</p