Figure 7. MG induces Hsp90 post-translational glycation in breast cancer cells.

(A) Immunoprecipitation of MG adducts on MG-treated MDA-MB-231 cells (300 µM, 6 hr) using a specific anti-argpyrimidine monoclonal antibody. Mouse immunoglobulins (IgG) were used as control. Total cell lysates (Input) and immunoprecipitates (IP) were immunoblotted for argpyrimidine, Hsp90 and Hsp27. (B) Under the same conditions as in A, MDA-MB-231 cell lysates were immunoprecipitated using anti-Hsp90. Inputs and IPs were immunoblotted using Hsp90 antibody and two specific antibodies directed against MG-adducts (argpyrimidine and hydroimidazolone MG-H). (C) Schematic representation of Hsp90 protein domains where hot spots (*) of endogenously and/or exogenously MG-modified residues are indicated. See also detailed amino acid sequence in Figure 7—figure supplement 1B. (D) Western blot analysis using the indicated antibodies on recombinant human Hsp90 (rhHsp90) incubated in presence of MG ± carnosine (10 mM) or 17-AAG Hsp90 inhibitor (1 µM) during 24 hr. (E) Hsp90 ATPase activity was decreased after incubation with MG or 17-AAG. This effect is efficiently blocked in presence of carnosine MG scavenger. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of five independent experiments. *p<0.05 and **p<0.01. (F) Co-immunoprecipitation of LATS1 and Hsp90 from MDA-MB-231 cells treated with MG 300 µM during 24 hr reveals a decreased interaction between the two proteins. Immunoblot data were quantified by densitometric analysis and normalized for β-actin. Numbers represent fold increase relative to the condition shown with bold number. All data are representative of three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.19375.020

Figure 7—figure supplement 1. MG induces Hsp90 post-translational glycation.

(A) MG treatment from low to high density did not affect HSP90 mRNA levels as assessed by qRT-PCR in the indicated cell lines. Data were analyzed using unpaired student’s t test and shown as the mean values ± SEM of three independent experiments. (B) MG-modifications represented on Hsp90α amino acid sequence. Underlined amino acids represent ATP binding site. Magenta and blue amino acids represent MG-modifications observed on recombinant and endogenous Hsp90, respectively. Red amino acids represent MG-modifications found in both conditions. (C) LATS1 expression is decreased in breast cancer cells treated with MG (300 µM) or 17-AAG (1 µM) during 24 hr. Immunoblot data were quantified by densitometric analysis and normalized for β-actin. Numbers represent fold increase relative to the condition shown with bold number. Western blot are representative of three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.19375.023