Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

<div><p>Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.</p></div

Characterization of HspB1, HspB5 and mutant HspB5 in HeLa cell clones.

<p>A) Cellular distribution of HspB1, HspB5 (wild type and mutant) and Hsp70 upon cell lysis. Neo, WT and R120G cells were lysed in the presence of 0.1% Triton X-100 and spun at 10,000×<i>g</i> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>. The levels of HspB1, HspB5 and Hsp70 present in the supernatant and pellet fractions were detected in immunoblots probed with the corresponding antibodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). Autoradiographs of ECL-revealed immunoblots are presented. Quantitative analysis of three independent experiments is presented in the adjacent figure. B) Effect of shRNA-mediated depletion of HspB1. WT and R120G cells were transiently transfected with control mismatch pSuperNeo-MsRNA27 (Mismatch: Ms) or pSuperNeo-ShRNA27 (ShB1) vector targeting HspB1 mRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). Two days after transfection, cells were analyzed in immunoblots probed with HspB1, HspB5 and actin antibodies. ShB1 transfected cells were also treated for the last 20 h before being analyzed with 0.5 µmol/l of MG132. Quantitative analysis of one particular experiment where the RNAi-mediated transient depletion of HspB1 was almost complete is presented in the adjacent figure. C) Analysis of HspB1 and HspB5 native sizes in Neo, WT and R120G cells. Cells were lysed as above and the 10,000×<i>g</i> cytosolic supernatant fractions were applied to Sepharose 6B gel filtration columns (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). The presence of HspB1 and HspB5 in pooled fractions eluted from the columns was detected in immunoblots probed with the corresponding antibodies. Autoradiographs of ECL-revealed immunoblots are presented. 29, 66, 150, 200, 443, 669 kDa are gel filtration markers. Exclusion size of the column is 2000 kDa. Brackets indicate fractions that were pooled for further immunoprecipitation analysis. Size population I is from WT cells and size population II is from R120G cells. D) Co-immunoprecipitation studies. a) Size population I from WT cells was immunoprecipitated with either anti-HspB1 (IPαB1) or anti-HspB5 antibody (IPαB5). Immunoprecipitated proteins-bound to proteinG-sepharose were washed in IPP150 buffer containing 150 mM NaCl before being processed for gel electrophoresis. After migration in SDS-PAGE, proteins were revealed in immunoblots probed with either anti-HspB1 or anti-HspB5 antibody. T: aliquot of cytosolic supernatant fractions before immunoprecipitation, IP: immunoprecipitated proteins, S: aliquot from supernatant after immunoprecipitation. b) Same as a) except that washes of the immunoprecipitated proteins were performed in IPP300 buffer containing 300 mM NaCl. c–d) same as a–b) but in this case analysis was performed with size population II from R120G cells. Autoradiographs of ECL-revealed immunoblots are presented.</p

Characterization of Neo, WT and R120G cells.

<p>A) Immunoblot analysis of total cellular extracts of Neo, WT and R120G HeLa cells. The levels of HspB1, HspB5, Hsp70, Hsp90, HspB6 and Actin were detected in immunoblots probed with the corresponding antibodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). B) Phase contrast analysis of Neo, WT and R120G. bar: 10 µm. Black arrows: perinuclear granules; white arrowheads: membranous ruffles. Analysis of the biggest dimension of cells (cell length) is presented in the adjacent figure. Mean, SD (standard deviation) and SEM (standard error of mean) of twenty different measurements are presented. C) Analysis of the number of cells in the cultures was from day 0 to days 1 and 2 (d1, d2). Values are means ± SEM of three independent experiments. One-way ANOVA within a time point analysis indicates statistically significant growth differences between Neo and WT and R120G cell lines, <i>*P</i><0.05. D) Immunofluorescence analysis. Neo, WT and R120G cells were processed for the immunofluorescence detection of HspB5, HspB1 and nuclei as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>. Bar: 10 µm. Cells were stained for HspB5 (red fluorescence), HspB1 (green fluorescence), nuclei (blue fluorescence) and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>. The fusion images (Merge) of WT and R20G cells are shown. Overlap and Pearson's coefficients are indicated. E) The graphs represent the fluorescence distribution of HspB1 (green; Ch1-1), wild type or mutant HspB5 (red; Ch1-2) and nucleus (blue; Ch1-3) of the section of WT or R120G cells shown in the green/red fusion images (Merge). #: areas where the co-localization of HspB1 and HspB5 (wild type or mutant) may not occur.</p

Analysis of the native size of phosphorylated HspB1 and HspB5.

<p>Non-treated as well as menadione-treated (60 µM, 2 h) Neo, WT and R120G cells were lysed and the S10,000×<i>g</i> supernatant fractions were applied to Sepharose CL-6B gel filtration columns as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>. The presence of HspB1, HspB5 and their different phosphorylated isoforms (HspB1: Ser15, Ser78 and Ser82/HspB5: Ser19, Ser45 and Ser59) were detected in immunoblots of the collected fractions probed with the corresponding antibodies. The corresponding native size of total HspB1 or HspB5 is shown (marked by a dark line surrounding immunoblots). Autoradiographs of ECL-revealed immunoblots are presented. 29, 66, 150, 200, 443, 669 are gel filtration markers. Exclusion size of the column is 2000 kD. Quantitative analysis of the presence of HspB1 and HspB5 in different size domains of the column is presented (Neo cells: a, b, c and d; WT cells: e, f and g; R120G cells: h, i and j). Results are presented as percentage of HspB1, HspB5 and their different phosphorylated isoforms in the different size populations in regard to their amount in the S10,000×<i>g</i> supernatants. Standard deviations are indicated from three independent experiments. <i>**P</i><0.01.</p

Quantitative analysis of the cellular distribution of phosphorylated HspB1 and HspB5.

<p>The data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g002" target="_blank">Fig. 2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g005" target="_blank">5</a> were used to calculate the percentage in regard to the total cellular content of HspB1, HspB5 and their different phosphorylated isoforms (HspB1: Ser15, Ser78 and Ser82/HspB5: Ser19, Ser45 and Ser59) in the different size populations (S10,000×<i>g</i>, gel filtration) and pellet fractions (P10,000×<i>g</i>). Size populations from gel filtration columns and color codes are the same as those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g005" target="_blank">Fig. 5</a>. Standard deviations are indicated from three independent experiments. <i>**P</i><0.01.</p

Enhanced oxidoresistance induced by wild type HspB5 and sensitivity mediated by the R120G mutation.

<p>Neo, WT or R120G cells were treated or not for different time periods with several concentrations of menadione. A) Crystal violet staining. The percentage of cell survival corresponded to the ratio of the relative absorbance of the different samples to that of untreated cells. Values are means ± SDM of three independent experiments. 2-way ANOVA indicates statistically significant differences in the survival to menadione treatment between Neo, WT and R120G cell lines, <i>*P</i><0.05, <i>**P</i><0.01. B) Clonogenic colony formation assay. The number of colonies was visually estimated. All experiments were performed in triplicate. C) Phase-contrast analysis of cell morphology. Before and after treatments, phase contrast analysis of the morphology of live cells was performed and photographs are presented. Bar: 10 µm. Black arrows: perinuclear granules; black arrowheads: filamentous bridges between cells; white arrowheads: membranous ruffles; white arrows: vacuoles. D) Immunoblot analysis of the level of HspB1, HspB5, Hsp70 and Hsp90 in menadione-treated Neo, WT and R120G cells.</p

Effect of HspB1 serine 15 phosphorylation on HspB1-HspB5 native size.

<p>A) Neo, WT and R120G cells were transiently transfected with pKS27S15G vector (phosphoserine 15 of HspB1 replaced by glycine, S15G vector; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and methods</a>) and HspB1 as well as HspB1/HspB5 native sizes were subsequently analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g005" target="_blank">Fig. 5</a>. Results obtained from R120G cells transiently transfected with the corresponding empty vector are also presented (control vector). Three size domains (I, II, and III) were defined in the R120G column fractions that corresponded to the h, i and j fractions indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g005" target="_blank">Fig. 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g006" target="_blank">6</a>. Autoradiographs of ECL-revealed immunoblots are presented. 29, 66, 150, 200, 443, 669 are gel filtration markers. Exclusion size of the column is 2000 kD. B) Quantitative analysis. The distribution of HspB1 between size domains I, II and III of transiently transfected R120G cells is shown and expressed as percentage of the HspB1 content in the 10,000×<i>g</i> supernatant loaded on the column. The following vectors were used: pKS control, pKS27S15G (phosphoserine 15 of HspB1 replaced by glycine), pKS2711-3D (the three phosphoserine sites of HspB1 replaced by aspartic acid) and pKS27wt (wild type HspB1). Standard deviations are indicated from three independent experiments.</p

Knock out of Hsp27 induces procaspase-3 proteasomal degradation without modulating the expression of its gene.

<p><i>A</i>, Total extracts of HeLa cell clones were analyzed in immunoblots incubated and revealed with caspase-3 antibody. Quantification of blots intensity was reported in a table. <i>B</i>, Each cell line was treated for 4 h with 0.25 µmol/L of staurosporine. Samples were analyzed in blot using anti caspase-3 antibody. <i>C</i>, Quantification of <i>procaspase-3</i> gene relative expression by qPCR experiments. <i>D</i>, Effect of proteolytic inhibitors. Samples were collected after being treated with MG132 or ALLN, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029719#pone-0029719-g003" target="_blank">Fig. 3</a>. Levels of procaspase-3 were revealed by western blot analysis.</p

Hsp27 knock down clones, enriched in G2/M cells, show decreased cell proliferation.

<p>Constitutive Hsp27 depleted clones of HeLa or MCF-7 were isolated as described in Materials and methods. <i>A</i>, HeLa or MCF-7 samples from independent Hsp27 depleted clones were collected and levels of Hsp27, 70 and 90 were analyzed by western blot. <i>B</i>, Immunoblot analysis of Hsp27, survivin, Bcl-2, Bcl_Xs/l, Bax, Bid and actin present in both HeLa and MCF-7 depleted clones. <i>C</i>, WST-1 test was performed each 12 h during five days. Proliferation index was determined and reported for each cell line (<i>p</i><0.01). <i>D</i>, Cell cycle study was performed by flow cytometry analysis after PI staining as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029719#pone.0029719-Moulin1" target="_blank">[26]</a>. Quantifications experiments were realized and reported in tables presented on the left of the flow cytometry analysis.</p

Transient down regulation of Hsp27 protein by shRNA sensitizes HeLa cells to cell death and promotes apoptosis.

<p><i>A</i>, pCI-Neo, pSuperNeo-ScRNA27 (Sc27), pSuperNeo-MsRNA27 (Ms27) and pSuper-ShRNA27 (Sh27) vectors were transiently expressed in HeLa cells. Samples were collected 48 h after transfection and Hsp27 level was analyzed by western blot. Level of actin was used as control. <i>B</i>, Sensitivity to cell death was determined by crystal violet analysis, as described in Materials and methods. Staurosporine, a pro-apoptotic kinase inhibitor, was used to induce apoptosis (<i>p</i><0.01). <i>C</i>, HeLa cells, treated for 4 h with different concentrations of staurosporine, were analyzed by western blot using an anti-caspase3 antibody.</p