Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding Ce ´line Guinez, Anne-Marie Mir, Yves Leroy, Rene ´ Cacan, Jean-Claude Michalski, Tony Lefebvre * UMR-CNRS 8576, UGSF, IFR 147, 59655 Villeneuve d’Ascq, France Received 4 July 2007 Available online 16 July 2007 Abstract Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against inju- ries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG 2 cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even if differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations sug- gesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Hsp70; Stress; O-GlcNAc; UDP-GlcNAc; Energy charge; Proteasome inhibition; Amino-acid analogue O-linked N-acetylglucosaminylation (O-GlcNAc) mod- ifies many key metabolic and structural proteins. It is a ubiquitous, abundant, and dynamic post-translational modification that consists in the linkage of a single N- acetylglucosamine to the hydroxyl groups of serine and threonine residues. O-GlcNAc versatility is regulated by a couple of enzymes, namely O-GlcNAc transferase (OGT) and O-GlcNAcase that associate in a complex, the O-GlcNAczyme [1]. UDP-GlcNAc, one of the OGT substrates, directly comes from glucose metabolism so much so O-GlcNAc ensues from UDP-GlcNAc avail- ability and extracellular glucose concentration [2]. While O-GlcNAc was described in 1984 [3], no example of a specific function associated with this post-translational modification has yet been clearly defined to date. In spite of everything, through the hundreds O-GlcNAc-bearing proteins identified, it seems that this glycosylation regu- lates proteins half-live and proteolytic processing [4,5], subcellular localisation [6], protein–protein interactions [7,8], DNA binding [9], and enzyme activity [10]. Numer- ous works report the protective, and more again, the beneficial effect of O-GlcNAc on protein stability and cell viability [4,11–17] but the mechanical process by which O-GlcNAc exerts its protection remains to be elu- cidate. Nevertheless, our group recently described that members of the 70-kDa Heat Shock Proteins (HSP70) family were endowed of an O-GlcNAc-binding property that we called HGBA for Hsp70-GlcNAc-Binding Activ- ity [18–20]. We have shown that HGBA increased when cells were exposed to hyperthermia [19] or when cells were depleted in glucose [20]. We speculate that Hsp70 is capable of protecting O-GlcNAc proteins by binding their sugar moiety through their lectin property, avoiding their subsequent proteasomal degradation. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.020 * Corresponding author. Fax: +33 3 20 43 65 55. E-mail address: tony.lefebvre@univ-lille1.fr (T. Lefebvre). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 361 (2007) 414–420