Hsp90 Is Cleaved by Reactive Oxygen Species at a Highly Conserved N-Terminal Amino Acid Motif

<div><p>Hsp90 is an essential chaperone that is necessary for the folding, stability and activity of numerous proteins. In this study, we demonstrate that free radicals formed during oxidative stress conditions can cleave Hsp90. This cleavage occurs through a Fenton reaction which requires the presence of redox-active iron. As a result of the cleavage, we observed a disruption of the chaperoning function of Hsp90 and the degradation of its client proteins, for example, Bcr-Abl, RIP, c-Raf, NEMO and hTert. Formation of Hsp90 protein radicals on exposure to oxidative stress was confirmed by immuno-spin trapping. Using a proteomic analysis, we determined that the cleavage occurs in a conserved motif of the N-terminal nucleotide binding site, between Ile-126 and Gly-127 in Hsp90β, and between Ile-131 and Gly-132 in Hsp90α. Given the importance of Hsp90 in diverse biological functions, these findings shed new light on how oxidative stress can affect cellular homeostasis.</p> </div

Identification of the site of cleavage within Hsp90.

<p>(A) Silver stained SDS-PAGE gel showing both C-term and N-term protein fragments of Hsp90β after 30 min incubation with A/M (2 mM/10 µM) supplemented with ADP (0.2 mM) and FeCl₃ (0.5 mM). Asterisks show the presence of contaminants. (B) Mass spectrometry analysis of cleaved Hsp90β. Deconvoluted spectra of the small N-term fragment gave a precise monoisotopic mass of 13777.1122 daltons. (C) The picture illustrates the location of the cleavage site occurring in Hsp90β. The IGQFGVGFYS motif corresponding to a conserved amino acid sequence in several Hsp90 proteins is highlighted in red. (D) Degradation of various Hsp90 client proteins in K562 cells treated with A/M (2 mM/10 µM).</p

Hsp90 cleavage by A/M requires the presence of ionic iron and ADP.

<p>K562 cell lysates (100 µg) were incubated for 1 h in the absence (Ctrl) or in the presence of A/M (2 mM/10 µM) supplemented with ADP (0.2 mM) and FeCl₃ (0.5 mM). Different concentrations of ADP (A), FeCl₃ (B) and MgCl₂ (C) were tested, as indicated. Hsp90 was detected with an anti-C terminus antibody from Santa Cruz Biotechnology (Hsp90 α/β, clone F-8).</p

Oxidative stress leads to Hsp90 cleavage and client protein degradation.

<p>(A) Time course of Hsp90 cleavage in K562 cells upon treatment with A/M (2 mM/10 µM). Hsp90 was detected with an anti-C terminus antibody from Santa Cruz Biotechnology (Hsp90 α/β, clone F-8). (B) Hsp90 cleavage in cells incubated for 2 hours with either A/M (2 mM/10 µM) or Glox (30 mM glucose/0.25 U/ml glucose oxidase), another H₂O₂-generating system (top left). Suppression of Hsp90 cleavage in cells preincubated for 1 h with 3 mM N-acetylcysteine (NAC) or 100 U/ml of catalase (CAT) and then exposed to A/M for 2 h (bottom left). Iron chelation by preincubating cells with deferoxamine (DFO) for 18 h decreased the cleavage induced by 2 h treatment with A/M (top right). Preincubation of cells for 1 h with 10 µM of N-term Hsp90 inhibitors, like geldanamycin (GA), 17-AAG (AAG) or radicicol (Rd), protected Hsp90 from the cleavage induced by 2 h treatment with A/M. Novobiocin (NB) at 1 mM did not protect (bottom right). Hsp90 was detected with the same antibody as in (A). (C) Cellular Hsp90 cleavage was reproduced in K562 cell lysates, in purified Hsp90 from HeLa cells, and in Hsp90 α and β recombinant proteins. K562 cells were incubated for two hours in the absence (Ctrl) or in the presence of A/M (2 mM/10 µM). K562 cell lysates (100 µg) were incubated for 1 h in the absence (Ctrl) or in the presence of A/M supplemented with ADP (0.2 mM) and FeCl₃ (0.5 mM). For the experiments with purified and recombinant proteins, we used 2 µg of Hsp90, incubation lasted 30 min and A/M (2 mM/10 µM) was supplemented with 0.2 mM ADP and 0.5 mM FeCl₃. Note that Hsp90 was detected with the same antibody as in (A) with the exception of recombinant Hsp90α that was detected with an anti-penta-His antibody.</p

Formation of Hsp90 protein radicals following oxidative stress.

<p>(A) Immuno-spin trapping blots showing the formation of nitrone adducts in Hsp90α (200 µg) incubated with DMPO (1 mM) and exposed to oxidative stress generated by Glox (60 mM glucose/1 U/ml glucose oxidase) supplemented with ADP (0.2 mM) and FeCl₃ (0.5 mM). Blank and Ctrl mean, respectively, that DMPO and Glox were omitted. (B) Same as (A) but with Hsp90β. (C) Proposed mechanism leading to oxidative polypeptide cleavage according to Stadman et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040795#pone.0040795-Berlett1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040795#pone.0040795-Stadtman2" target="_blank">[19]</a>.</p

Supplementation with <i>Lactobacillus reuteri</i> 100-23 and <i>Lactobacillus gasseri</i> 311476 mixture restores the lactobacilli levels.

<p><b>A–D</b>. <i>Lactobacillus</i> spp., <i>L. reuteri</i>, <i>L. jonhsonii/gasseri</i> and <i>L. murinus/animalis</i> levels in control mice (CT), in mice receiving lactobacilli (Lrg), in mice transplanted with BaF3 cells (BaF3) and in mice transplanted with BaF3 cells and receiving lactobacilli (BaF3-Lrg). <b>E</b>. Daily food intake. N = 7–8. Data with different superscript letters are significantly different (p<0.05). For the food intake, n = 4 and *p<0,05 BaF3 and BaF3-Lrg mice versus CT and Lrg mice.</p

Changes in gut microbiota composition occur in mice transplanted with BaF3 cells.

<p><b>A</b>. DGGE profiles of the bacterial DNA isolated from the cecal content. Each profile corresponds to one animal. UPGMA dendogram with Dice coefficient. <b>B</b>. Levels of total bacteria, <i>Bacteroides</i> spp. and <i>Lactobacillus</i> spp. (quantified with two different sets of primers). <b>C</b>. <i>Lactobacillus johnsonii/gasseri, Lactobacillus murinus/animalis</i> and <i>Lactobacillus reuteri</i> levels. <b>D</b>. Relative proportions of each <i>Lactobacillus</i> species, expressed as a percentage of the <i>Lactobacillus</i> spp. levels (second primer set). N = 8, *p<0.05 vs. control. Percentages of each species of lactobacilli are significantly different (p<0.05 with Student’s t-test) between control mice (CT) and mice transplanted with BaF3 cells (BaF3 mice).</p

Transformed mouse proB BaF3 cells infiltrate the liver and spleen.

<p><b>A</b>. Liver and spleen weights. N = 8. *p<0.05 vs. control. <b>B</b>. Bcr-Abl mRNA levels in liver and spleen, ND: not detected. <b>C.</b> Histological analysis of liver tissue (hematoxylin-eosin staining). Bar  = 50 µm. White arrows indicate BaF3 cells.</p

Supplementation with <i>Lactobacillus reuteri</i> 100-23 and <i>Lactobacillus gasseri</i> 311476 mixture reduces muscle atrophy markers.

<p><b>A–D</b>. Atrophy marker expression (Atrogin-1, MurF1, LC3 and Cathepsin L) measured in the gastrocnemius muscle of control mice (CT), mice receiving lactobacilli (Lrg), mice transplanted with BaF3 cells (BaF3) and mice transplanted with BaF3 cells and receiving lactobacilli (BaF3-Lrg). <b>E–H</b>. Atrophy marker expression (Atrogin-1, MurF1, LC3 and Cathepsin L) measured in the tibialis muscle. <b>F</b>. Muscle weights. N = 7–8. Data with different superscript letters are significantly different (p<0.05).</p

Body weight gain, leukemia- and fat mass-related parameters, blood glucose and lipids.

<p>N = 7–8, data with different superscript letters are significantly different (p<0.05). ND  =  not detected.</p