Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins Benjamin Gibert1,6*, Be´ ne´ dicte Eckel1,4, Lydie Fasquelle1,2, Maryline Moulin1,3, Frantz Bouhallier5, Vincent Gonin4, Gregory Mellier1, Ste´ phanie Simon 1 , Carole Kretz-Remy 1 , Andre´ -Patrick Arrigo1,6, Chantal Diaz-Latoud1,4* 1 Centre de Ge´ne´tique Mole´culaire et Cellulaire, CNRS UMR5534, Universite´ Lyon 1, Universite´ de Lyon, Villeurbanne, France, 2 Inserm U583, Institut des Neurosciences, Hoˆ pital Saint Eloi, Montpellier, France; Universite´ Montpellier 1, Montpellier, France, 3 Department of Biochemistry, La Trobe University, Victoria, Australia, 4 INSERM U664, Universite´ Lyon 1, Universite´ de Lyon, Villeurbanne, France, 5 Institut de Ge´nomique Fonctionnelle de Lyon, Universite´ Lyon 1, ENS Lyon, CNRS, UMR5242, INRA, UMR1288, Lyon, France, 6 Apoptosis, Cancer, and Development Laboratory, CRCL UMR INSERM U1052-CNRS UMR5286, Universite´ de Lyon, Centre Le´on Be´rard, Lyon, France Abstract Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions. Citation: Gibert B, Eckel B, Fasquelle L, Moulin M, Bouhallier F, et al. (2012) Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins. PLoS ONE 7(1): e29719. doi:10.1371/journal.pone.0029719 Editor: Harm Kampinga, University Medical Center Groningen, University of Groningen, The Netherlands Received September 12, 2011; Accepted December 2, 2011; Published January 4, 2012 Copyright: 2012 Gibert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a research grant from the Comite´ du Rhoˆ ne of la Ligue Contre le Cancer and by the Rh oˆne-Alpes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chantal.diaz@univ-lyon1.fr (CD-L); benjamin.gibert@lyon.unicancer.fr (BG) Introduction Hsp27, also called HspB1, is a member of the small heat shock family of proteins that are characterized by their conserved C- terminal a-crystallin domain [1]. This oligomeric phosphoprotein bears an ATP-independent chaperone activity [2]. Hsp27 is also known for its anti-apoptotic activities [3] that appear of complex nature because of the dynamic and specific changes in the structural organization of this protein in response to various stimuli. Hence, multiple strategies appear to be set up by Hsp27 to interfere with apoptotic processes [4]. Indeed, Hsp27 can interact with several components of the apoptotic machinery. It interferes with apoptotic receptor like CD95-Fas/Apo1 by sequestering Daxx, a polypeptide crucial for death signal transduction [5]. Hsp27 also interacts with cytochrome c, once it is released from mitochondria, hence preventing apoptosome formation [6]. A binding to procaspase-3 has been observed that prevents the cleavage into active caspase-3 [7]. Hsp27 also interferes with targets upstream of mitochondria, as for example F-actin, hence preventing its disruption and aggregation. This delays the accumulation of cytochrome c in the cytosol and subsequently reduces caspases activation [8]. Hsp27 is phosphorylated by the p38MAPK/MAPKAPK2 pathway and promotes the activation of the pro-survival serine/threonine kinase Akt [9,10]. Hsp27 is well referenced as a therapeutic target in cancer [11] since its increased expression in several types of tumor cells correlates with increased aggressiveness, lack of response to therapies and bad prognostic for patients [12,13]. For example, Hsp27 expression enhances the resistance to chemotherapeutic drugs like cisplatin, adriamycin and bortezomib [14,15] and protects against radio-therapeutic radiations, probably as a consequence of its ability to act as an anti-oxidant polypeptide [16]. Thus, targeting Hsp27 level by antisense strategies sensitizes cells to c-rays [17]. The molecular chaperone distinctiveness of Hsp27 implicates that this constitutively expressed protein could directly interfere with several target proteins and regulates numerous cellular processes. In this regard, one member of the heat shock protein family, Hsp90, is well characterized to interact with an important number of client proteins implicated in cell cycle regulation, signal transduction or gene transcription [18,19]. By doing so, Hsp90 promotes the stability and activity of polypeptides by controlling, through its chaperone activity, their folding. These studies have been facilitated by the existence of specific chemical inhibitors, i.e benzoquinone ansamycin geldanamycin and its less toxic deriva- tive 17-allylamino-17-demethoxygeldanamycin (17-AAG), which bind Hsp90 ATPase box and knock out the chaperone activity. This disrupts Hsp90 interaction with client proteins that are subsequently degraded through the ubiquitin-dependent protea- somal pathway [20]. Such a mechanism is well referenced for Hsp90 but less documented for other chaperones. However, reports have already mentioned decreased levels of procaspase-3, PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29719