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RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing
Published on: September 27, 2016
Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells
Casper G Schalkwijk1, Jan van Bezu, Roel C van der Schors
1Department of Clinical Chemistry, Vrije Universiteit Medical Center, Amsterdam, The Netherlands. c.schalkwijk@intmed.unimaas.nl
Abstract:
In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.
Insights
High glucose conditions increase methylglyoxal, leading to elevated argpyrimidine adducts in endothelial cells. This modification of heat-shock protein 27 (Hsp27) may impair cell function in diabetes.
Area of Science:
- Biochemistry
- Cell Biology
- Endocrinology
Background:
- High glucose levels in diabetes promote the formation of advanced glycation endproducts (AGEs).
- Methylglyoxal is a key precursor to AGEs, particularly in endothelial cells.
- Specific AGE adducts, like argpyrimidine, are implicated in diabetic complications.
Purpose of the Study:
- To investigate the role of methylglyoxal in AGE formation under high glucose conditions.
- To identify specific AGE adducts and modified proteins in endothelial cells.
- To explore the functional consequences of AGE modification on endothelial cells.
Main Methods:
- Endothelial cells were cultured under varying d-glucose concentrations (5 mM vs. 30 mM).
- Levels of methylglyoxal and specific AGEs (argpyrimidine, N(e)-(carboxymethyl)lysine, N(e)-(carboxyethyl)lysine) were quantified.
- Protein identification of modified targets was performed using mass spectrometry after in-gel digestion.
Main Results:
- High glucose significantly increased intracellular methylglyoxal and argpyrimidine levels.
- Argpyrimidine levels were also elevated by glyoxylase I inhibition (HCCG) and PPARgamma activation (troglitazone).
- Heat-shock protein 27 (Hsp27) was identified as a major argpyrimidine-modified protein, and its modification correlated with decreased endothelial cell proliferation.
Conclusions:
- Methylglyoxal-derived argpyrimidine modification of Hsp27 occurs in endothelial cells under hyperglycaemic conditions.
- This modification is associated with reduced endothelial cell proliferation, potentially contributing to diabetic vascular dysfunction.
- Targeting methylglyoxal or its adducts may offer therapeutic strategies for diabetic complications.
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