Effects of protein-secretion inhibitors on Hsp60 secretion by tumor cells.

<p><b>A</b>) Hsp60 and Hsp70 detected by Western blotting in: (<b>a</b>) immunoprecipitates from conditioned media from untreated (Unt) and inhibitor-treated H292 tumor cells; and (<b>b</b>) whole-cell lysates from H292 cells. The inhibitors are listed on top of the respective lanes. Histograms to the right represent the levels of the Hsps in immunoprecipitates determined in three separate experiments as mean percentages +/− SD of arbitrary units (AU) obtained with NIH image J 1.40 analysis software. * and ** significantly different from untreated control, p<0.005 and p<0.001, respectively. The two inhibitors (listed below the bars) significantly decreased secretion of Hsp60 and Hsp70. Also, the data from whole-cell lysates show that the protein-secretion inhibitors had no detectable effect on Hsp levels inside the cells. <b>B</b>) Hsp60 levels secreted by the H292 tumor cells before and after exposure for 1 hour, followed by a 4 hours recovery period, to protein-secretion inhibitors measured by ELISA in: (<b>a</b>) conditioned media; and (<b>b</b>) exosomes. Histograms represent Hsp60 levels expressed as pg of protein normalized for mL normalised for 10⁶ cells. Data represent mean +/− SD of three different experiments in duplicate. * Significantly different from untreated control, p<0.005. The results, which are in agreement with those obtained by Western blotting, show that the inhibitors tested significantly reduced secretion of Hsp60 by the H292 tumor cells.</p

Hsp60 is integrated in the membrane of exosomes from tumor cells. A–C.

<p>Illustrative data on the exosome preparations utilized in this work. <b>A</b>, Transmission electron microscopy demonstrates that the dimension of isolated vesicles is equal to, or smaller than 100 nm, which is consistent with exosomes. Bar: 100 nm. <b>B</b> and <b>C</b>, acetylcholinesterase (AChEase) and ATPse enzymatic activities, respectively, typical of isolated exosomal vesicles, compared to control (conditioned culture medium). In B, the solid line represents data from exosomes, and the broken line represents results from conditioned culture medium. Vertical axis, AChEase activity in arbitrary units (AU), reflecting 412 nm absorbance; horizontal axis, time of reaction in minutes. In C, 1, marker (positive control); 2, conditioned culture medium; 3, exosomes. <b>D.</b> Treatment with sodium carbonate alone (lane 3, asterisk), or in association with proteinase K buffer (lane 4), does not remove Hsp60 from the exosomes, whereas treatment with Proteinase K does (lane 2). Lane 1, untreated exosomes (positive control).</p

Golgi involvement in Hsp60 secretion from tumor cells. A.

<p>TEM<b>-</b>Immunogold shows Hsp60 (black dots) in tumor cells, including in the Golgi (framed by a dashed rectangle). The arrows show the plasma membrane and arrowheads indicate Hsp60 in it. Bar: 100 nm. <b>B.</b> Extracellular levels of Hsp60 decrease after Brefeldin A (BFA) treatment of the tumor cells, as measured in immunoprecipitates from the conditioned medium (left upper panel). In contrast, Hsp70 levels are not influenced by BFA treatment (left lower panel). Right hand panel: histograms showing densitometric measurements of the Western blots to the left. UT, untreated; A.U.: arbitrary units; asterisk indicates p<0.005. <b>C.</b> ELISA results demonstrate a reduction of Hsp60 levels in the conditioned culture medium from tumor cells after BFA treatment. UT: untreated. <b>D.</b> ELISA results show a reduction of Hsp60 levels in exosomes after BFA treatment of the tumor cells. UT: untreated. Asterisks in C and D, p<0.05. Error bars represent SD.</p

Blue Native Polyacrylamide Gel Electrophoresis: Hsp60 at various concentrations.

<p>Blue Native PAGE (4–16%) image of naïve Hsp60 in the concentration range 1.9–4.8 µM. The pattern reveals that the protein exists in two oligomeric forms independently of the concentration.</p

Size exclusion chromatography results.

<p>Size exclusion chromatography experiments on the naïve Hsp60 at different concentrations (0.8 µM: red, 1.6 µM: black, 2.2 µM: magenta, 6.4 µM: green, 16.0 µM: turquoise) compared with GroEL at 7.0 µM (blue) and BSA (dotted line). The vertical line drawn across the Hsp60 peak helps to highlight the independency of the retention time from protein concentration. The value of the retention time is consistent with that expected for heptameric and tetradecameric species in equilibrium.</p

SAXS for GroEL and Hsp60: Guinier approach.

<p>SAXS profiles of naïve Hsp60 at 23.8 (green) and 79.5 µM (blue). Experimental curves are scaled for the sake of clarity. Continuous lines correspond to the theoretical fitting obtained by the Guinier approach, as reported in Material and Methods, SAXS. The gyration radius extrapolated by fitting procedure suggests, at both concentrations, the simultaneous presence of oligomeric structures in equilibrium.</p

SAXS profiles of GroEL and Hsp60.

<p>SAXS profiles of GroEL (red) and naïve Hsp60 (green). GroEL is at c = 52.5 µM, while Hsp60 is at c = 79.5 µM. Experimental curves are scaled for the sake of clarity. SAXS spectrum for Hsp60 is quite different from that of GroEL, which corresponds to a more well-defined structure.</p

Static light scattering characterization.

<p>Scattered light intensities from solutions of naïve Hsp60 protein at different concentrations, expressed in terms of the Rayleigh ratio <i>R_90°</i> at 18.7 µm⁻¹ normalized by the concentration values <i>c</i>. Together with the total scattered intensity (empty circles), the intensity contribution by species with diameter size lower than 70 nm (filled circles) is also reported. The lines represent the dependence of <i>R_90°/c</i> on concentration predicted for Hsp60 protein totally assembled in tetradecamers (solid) or heptamers (dashed). The experimental values of intensity scattered by Hsp60 species always fall between those predicted for totally tetradecameric or heptameric populations.</p

Dynamic light scattering characterization.

<p>Dynamic light scattering characterization of the naïve Hsp60 at different concentrations (0.8 µM: black, 6.4 µM: green, 16.0 µM: turquoise, 27.0 µM: orange) compared with GroEL at 7.0 µM (blue). (A) Normalized intensity autocorrelation functions g⁽²⁾(t). (B) Number-weighted distribution functions P_N of the <i>z</i>-average hydrodynamic diameter D_H obtained by the analysis of the autocorrelation functions. At each concentration, the hydrodynamic diameter of Hsp60 is always compatible with that of heptamer/tetradecamer species.</p

Blue Native Polyacrylamide Gel Electrophoresis: Hsp60 at various concentrations.

<p>Blue Native PAGE (4–16%) image of naïve Hsp60 in the concentration range 1.9–4.8 µM. The pattern reveals that the protein exists in two oligomeric forms independently of the concentration.</p