1A_01_S

Hsp27 (HspB1) as a therapeutic target

Arrigo André-Patrick

Stress, Chaperons and Cell Death Laboratory, CGMC, CNRS UMR 5534, Claude Bernard University LYON 1, 16 Rue Dubois, Bat. Gregor Mendel, 69622 Villeurbanne, France, arrigo@univ-lyon1.fr

Human Hsp27(HspB1) is a molecular chaperone which is constitutively expressed in several mammalian cells, particularly in pathological conditions. This protein has functions as diverse as protection against toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In addition, it has anti-apoptotic properties and is tumorigenic when expressed in cancer cells. Hsp27(HspB1) has implications, either positive or deleterious, in pathologies such as neurodegenerative diseases, asthma, and cancers. Moreover, mutations in hsp27 gene have been detected which are responsive of hereditary motor neuropathies. Hsp27(HspB1) is therefore an active determinant in health and disease and not just a passive storage device. Approaches as well as preliminary results towards therapeutic strategies aimed at modulating the expression and/or the activities of Hsp27(HspB1) will be presented.

1A_02_S

HspB8 forms with Bag3 a chaperone complex stimulating macroautophagy: possible involvement of sHsp in protein quality control

Jacques Landry

Centre de recherche en cancérologie de l’Université Laval, L’Hôtel-Dieu de Québec, Québec, Canada, G1R 2J6, jacques.landry@med.ulaval.ca

Intracellular protein aggregation can occur upon proteotoxic stress or genetic mutations and represents a major threat in the crowded environment of the cell. The Hsp70/Hsp90 proteins and their associated co-chaperones form a protein quality control system which can recognize such proteins and either assist them in renaturation or target them for degradation. Proteins from the small Hsp family, and in particular HspB1 (Hsp27) and HspB8, may similarly act as molecular chaperones as evidenced by their association with human diseases featuring protein conformation defects. Diverse types of activities are associated with each of these proteins. On one hand, HspB1 (Hsp27) but not HspB8 potentiates the renaturation of damaged proteins in the cells. In this activity, HspB1 recognizes the substrate and then recruits a renaturating machinery likely involving Hsp70 family members. On the other hand, HspB8, but not HspB1, forms in cells a stable protein complex with Bag3, a Bag family member previously identified as a Hsp70 co-chaperone. In association with Bag3 but independently Bag3 binding to Hsp70, HspB8 efficiently limits the accumulation of aggregation-prone protein substrates by targeting them to destruction by macroautophagy, a lysosome-based protein degradation system capable of degrading large structures and insoluble protein aggregates. In this complex HspB8 likely acts as a molecular chaperone responsible for the recognition the damaged substrates whereas Bag3 is responsible for recruiting and stimulating the macroautophagy machinery. Accordingly, HspB1 engineered to interact with Bag3 can also target unstable proteins to degradation. Hence HspB proteins can, like Hsp70/Hsp90, function in protein quality control by recognizing protein substrates, the fate of which being determined by associated co-chaperones that are specific to individual chaperones.

1A_03_S

Recovery of macromolecular synthesis after stress: the role of small heat shock protein

Nicolette H. Lubsen, N.Lubsen@science.ru.nl

Cells stressed by heat downregulate protein synthesis. During recovery from heat stress protein synthesis slowly recovers. In the presence of either ?B-crystallin or Hsp27, both small heat shock proteins, the rate of recovery is enhanced. Using fluorescence recovery after photobleaching we showed that Hsp27, but not ?B-crystallin, increased the pool of mobile stress granule-associated EGFP-eIF4E in heat shocked cells. Hsp27 also partially prevented the sharp decrease in the pool of mobile cytoplasmic EGFP-eIF4G, supporting other evidence for a direct interaction between eIF4G and Hsp27. sHsps did not prevent the phosphorylation of eIF2? by a heat shock, but promoted dephosphorylation during recovery. Blocking the endogenous heat shock response by expressing a dominant negative HSF1 mutant during recovery from a heat shock slows the rate of recovery of protein synthesis and blocks the restorative effect of sHsps, showing that sHsps need to cooperate with other Hsps of which the synthesis is induced by heat shock. Translational recovery 24 hours after heat shock does not differ between cells expressing dnHSF1 and control cells. However, if cells express dnHSF1 as well as the C-terminal fragment of GADD34, which causes constitutive dephosphorylation of eIF2?, translation does not recover. These data show that two partially redundant pathways are involved in translational recovery from a heat shock.

1A_04_S

Robert M. Tanguay, Genevieve Morrow, Hyun-Ju Kim, Sébastien Michaud

Small HSP are involved in the refolding and/or disposal of protein aggregates, a feature of many age-associated diseases. In Drosophila melanogaster, there are 4 main small Hsps each residing in a different intracellular compartment. Targeting the expression of the mitochondrial Hsp22, to different cell types can increase lifespan by more than 30%. Long-lived flies expressing Hsp22 in motorneurons have an increased resistance to oxidative stress and maintain their locomotor activity longer. Over expressing the cytosolic Hsp23 in a pan-neuronal fashion (transgenic GAL4/UAS system) also increases longevity by 15%. Conversely, a strain carrying an insertion in the promoter of hsp23 which downregulates its expression in specific cells of the embryonic CNS and in adults has a decreased lifespan. The action of these chaperones on lifespan likely involves different pathways as suggested by the longevity curves, and their respective site and developmental pattern of expression. Microarrays analysis was used to unveil the mechanisms involved in lifespan. The transcriptional changes brought by Hsp22 overexpression occur early in adulthood and are associated with genes involved in energy metabolism, protein biosynthesis, and protein folding. The relation between the insulin/IGF signaling pathway and the heat shock response has also been examined using flies with mutations in the heat shock factor HSF and dFOXO. Altogether, these results confirm a beneficial role of the expression of small chaperones and corroborate the pivotal role of the nervous system and the insulin/IGF pathway in the ageing process. Supported by CIHR (Canada) and the EU 6^th Framework Programme MiMage.

1A_05_S

Inhibition of apoptotic cell death by a novel 16.2 kD heat shock protein via Hsp90 mediated lipid rafts stabilization and Akt activation pathway

Balazs Sumegi¹, Szabolcs Bellyei^1,2, Eva Pozsgai¹, Andras Szigeti^1,2, Arpad Boronkai^1,2, Ferenc Gallyas Jr.¹

Correspondence to: Balazs Sumegi, PhD, DSci, Department of Biochemistry and Medical Chemistry, University of Pécs, 12 Szigeti Street, Pécs H-7624, Hungary. Tel: +36-72-536-276 Fax36-72-536-277 e-mail: balazs.sumegi@aok.pte.hu

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. While over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase-Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase - Akt cytoprotective pathway.

1A_06_S

Modulation of the Chaperone-like Function of Small Heat Shock Proteins by Methylglyoxal

Ram H. Nagaraj, Ashis Biswas, Manjunatha Bhat. Case Western Reserve

University and Cleveland Clinic Foundation, Cleveland, OH, USA

Alpha-crystallin and Hsp27 belong to the family of small heat shock proteins. They are stress proteins and play an important role in preventing protein aggregation and cell death by external stress. Methylglyoxal is an ubiquitous alpha-dicarbonyl compound produced from the triose phosphate intermediates of glycolysis. It reacts rapidly with arginine, cysteine and lysine residues in proteins and chemically modifies them to form stable adducts. We have studied the effect of methylglyoxal modification on the chaperone-like function of alpha-crystallin and Hsp27. Methylglyoxal modification of these two stress proteins increased their chaperone function on a concentration-dependent manner. Specific arginine modification to argpyrimidine was found to be responsible for the enhanced chaperone function. Site-directed mutagenesis of methylglyoxal-modifiable arginine residues to alanine mirrored the effects of methylglyoxal. Introduction of additional guanidino groups abolished the chaperone-like function of alphaA-crystallin but subsequent modification by methylglyoxal not only revived the chaperone-like function but also made it better than the unmodified protein. Modification of both substrate proteins and alphaA-crystallin by methylglyoxal further enhanced resistance to protein aggregation by thermal and chemical stress. These results suggest that physiological dicarbonyl methylglyoxal promotes the chaperone function of alphacrystallin and Hsp27 and prevents aggregation of proteins and thus may play a vital role in cell response to stress in health and disease.

1A_07_S

Two small heat shock proteins of a fission yeast function in different manner to cope with wide range of temperatures and various denatured proteins

Masafumi Yohda^1*, Chika Sugino¹, Maya Hirose¹, Ryo Iizuka¹, Masafumi Shimizu², Shun-ichi Kidokoro³, Noriyuki Ishii⁴,

*Corresponding author, yohda@cc.tuat.ac.jp

There exist two small heat shock proteins (SpHsp15.8 and SpHsp16.0) in the fission yeast, Schizosaccharomyces pombe (S. pombe). At the elevated temperatures, both SpHsp15.8 and SpHsp16.0 dissociate into small oligomers and then interact with denatured substrate proteins. SpHsp16.0 exhibited clear enthalpy change for denaturation at over 60 oC in differential scanning calorimetry. In addition, there was another small enthalpy change at about 50 oC, which is likely to correspond to oligomer dissociation. The oligomer dissociation and interaction with denatured protein of SpHsp15.8 and SpHsp16.0 were analyzed by fluorescence polarization analysis (FPA). Both sHsps exhibited temperature dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of SpHsp15.8 oligomer started to occur at about 35 oC and proceeds gradually. On the contrary, SpHsp16.0 oligomer was stable up to about 45 oC, but dissociate to small oligomers abruptly at the temperature. Interaction between sHsps and denatured CS at the elevated temperature was also examined by FPA. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, but SpHsp15.8 in the large complex in contrast. The results suggest that S. pombe, utilizes two sHsps to cope with wide range of temperature and various denatured proteins.

1B_01_S

Functional interplay among the major chaperone machines of e. coli

Ronald S. Ullers¹, Debbie Ang¹, Françoise Schwager¹, Pierre Genevaux², and Costa Georgopoulos¹

1Département de Microbiologie et Médecine Moléculaire, Centre Médical Universitaire, 1, Rue Michel-Servet, CH-1211 Geneva, Switzerland; and ²Laboratoire de Microbiologie et Génétique Moléculaires, Institut de Biologie Cellulaire et de Génétique, Centre National de la Recherche Scientifique, Université Paul-Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex 09, France

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase’s (PPIase) activity. Furthermore, in contrast to the previously known synergistic action of TF and the DnaK/DnaJ chaperone machine above 30°C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly somehow interferes with the process of cotranslational protein targeting to the Sec translocon. Various models to explain our results (occasionally seemingly contradictory) will be offered.

1B_02_S

Role of Hsp110 in the functional network of Hsp70 chaperones

Jocelyne Fiaux, Claes Andréasson, Heike Rampelt, Heather Sadlish, Matthias Mayer and Bernd Bukau

Heidelberg, Germany, bukau@zmbh.uni-heidelberg.de

Eukaryotic cells express Hsp110 proteins that constitute a diverged branch of the Hsp70 molecular chaperone superfamily. Recently, both the yeast Hsp110 member Sse1p and the mammalian orthologue Hsp105 were found to act as potent nucleotide exchange factors for cytosolic Hsp70 chaperones. We set out to characterize the steps of the nucleotide exchange cycle of the yeast Hsp70, Ssa1p, catalyzed by Sse1p using H/D exchange and mass spectrometry. The mechanism was found to involve formation of a stable but nucleotide-sensitive complex. Furthermore, Sse1p itself displays nucleotide binding and hydrolysis and adopts conformations dependent on the nucleotide binding status. Interestingly, nucleotide binding by Sse1p appears to regulate its activity as a nucleotide exchange factor. We also used H/D exchange and mass spectrometry to map the interaction surface of the Sse1p-Ssa1p complex. Based on these results, we can now propose a detailed model for the Sse1p-catalyzed nucleotide exchange cycle.

1B_03_S

Networks of Hsp70 and J-protein Molecular Chaperones

Elizabeth A. Craig, Alison Meyer and Chandan Sahi

Department of Biochemistry, University of Wisconsin, Madison WI, 53706, USA ecraig@wisc.edu

Highly conserved molecular chaperones function in a wide variety of cellular processes, including protein folding, translocation of proteins across membranes and remodeling of protein complexes. J-proteins are obligate partners of Hsp70s that act via their J-domains to stimulate Hsp70’s ATPase activity, stabilizing their interactions with client proteins. In addition many J-proteins contain additional domains, some of which are capable of binding client proteins and allowing J-proteins to “deliver” them to their partner Hsp70. Both Hsp70s and J-proteins are encoded by large multigene families. Our results indicate that certain Hsp70s and J-proteins have evolved to function in specialized cellular processes and/or have “nontraditional” functions. For example, Ssz1 forms a stable heterodimer with the ribosome-associated J-protein Zuo1 and is required for Zuo1’s efficient stimulation of the ATPase activity of its partner Hsp70 Ssb. Jjj1, is an example of a specialized J-protein. It plays an important role in the biogenesis of 60S ribosomal subunits, likely facilitating the dissociation of factors from preribosomes to generate subunits competent for translation. In contrast, other J-proteins are multifunctional. The most abundant J-protein of the yeast cytosol, Ydj1, functions in multiple cellular processes. Surprisingly, however, expression of only a J-domain at normal levels is capable of rescuing the severe growth defect caused by the absence of Ydj1. Thus many functions carried out by this highly conserved and complex J-protein require only the capacity to stimulate Hsp70s ATPase activity, not the “delivery” of client proteins.

1B_04_S

Hsp110 protein chaperone function in yeast

Kevin Morano

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors, and in yeast Sse1 is required for Hsp90 chaperoning. We demonstrate that Sse1 forms high affinity heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones, and that ATP binding to Sse1 is required for binding to Hsp70s. The nucleotide binding domains (NBD) of both Sse1/2 and the Hsp70s dictate interaction specificity, and are sufficient to mediate heterodimerization with no discernable contribution from the peptide binding domains (PBD). However, the PBD is required for NEF activity. To better understand the roles of the Hsp110 chaperones, we are investigating the participation of Sse1 in cellular processes requiring Hsp70. To that end, we have generated a novel temperature sensitive allele of SSE1 that should shed light on the essential functions of this intriguing chaperone family.

1B_05_S

Evolution and diversity of human Hsp70: implications for health and disease

Luciano Brocchieri¹, Everly Conway de Macario², Alberto J. L. Macario²

1University of Florida, Department of Molecular Genetics and Microbiology and UF Genetics Institute, Gainesville, FL, USA; ²Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USA.

Defective chaperones are implicated in disease, the chaperonopathies, and senescence. Defective chaperone identification and elucidation of pathogenetic role in disease and ageing requires full gene identification in the genome; characterization of genes and proteins in silico, in vitro and in vivo; and clinicopathological studies. We applied a series of complementary bioinformatics and evolutionary methods (chaperonomics) to the study of human Hsp70, and identified 47 loci encoding Hsp70-like sequences: 16 were functional genes, encoding proteins conserved at the N-terminal but not at the C-terminal domain, of which eight encoded typical Hsp70s (65-80kDa) with nucleotide-binding and substrate-binding domains, two encoded products lacking most or all of the C-terminal domain, and six encoded heavier proteins (> 81 kDa) with atypical C-terminal domains. Also, 31 hsp70-related pseudogenes were identified. Evolutionary analyses showed that ER-residing Hsps evolved by duplication while the six typical genes whose products reside in the cytosol/nucleus, and the majority of the pseudogenes, originated from retrotransposition of one gene, HSPA8. These evolutionary processes generated a group of genes with wide diversity further amplified by the occurrence of multiple mRNA variants and protein isoforms. The variety of the Hsp70 proteins is reflected in the diversity of their patterns of expression and localization in an assortment of tissues, cell types and sub-cellular compartments, through the stages of development and ageing. Consequently, the pathogenetic impact of defective Hsp70s should be widespread.

1B_06_S

Heat shock cognate protein HSC70 as a regulator of the Activin/Nodal/TGF-ß-Smad2 signalling pathway during zebrafish development

Michiaki Yamashita, Misako Hojo, and Takeshi Yabu

National Research Institute of Fisheries Science, Fukuura, Yokohama 236-8648, Japan, mic@affrc.go.jp

The cytosolic heat shock protein 70 (HSP70) and heat shock cognate protein 70 (HSC70) are thought to combine to function as a molecular chaperone. The protein binds transiently to nascent polypeptides and unfolded proteins, and prevents intramolecular and intermolecular interactions, which can result in misfolding or aggregation. Precise control of Nodal proteins, which are members of the transforming growth factor-ß (TGF-ß) superfamily, has been identified as a key endogenous mesoderm inducer in vertebrates. In the present study, we report that the HSC70 molecular chaperone promotes the mesoderm induction activities of Nodal signalling. To study the biological function of HSC70, the endogenous expression of HSC70 was knocked down by the injection of a specific antisense morpholino oligonucleotide (HSC-MO). The HSC-MO-injected embryos showed reduced phosphorylation of Smad2 by Nodal signalling, and this phenotype was rescued by co-injection of the HSC70 protein. However, the HSP70 mutant that lacked the C-terminal tetrapeptide (EEVD) motif showed no activity upon induction of Nodal signalling. The Activin type IIB receptor (ActRIIB) was immunoprecipitated with HSC70, and its autophosphorylation was enhanced in the presence of HSC70, which suggests that HSC70 binds directly to the receptor and modulates its activity. Therefore, HSC70 plays essential roles in the formation and activation of the type IIB receptor upon Nodal signalling.

1C_01_S

Multi-site post-translational modifications and functional interplay of HSF1 and HSF2

1C_02_S

HSF1 activation and the control of apoptosis in chemoresistant cancers

G. Belardo, A. Rossi, S. Ciafre’, A. Ciucci, P. Gianferretti, S. Roberts and M.G. Santoro

Department of Biology, University of Rome Tor Vergata and Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy; Department of Chemistry, University of Manchester, Manchester, UK.

Activation of the heat shock response (HSR) via HSF1 contributes to preserve cellular function and homeostasis under stress conditions, and to establish a cytoprotective state in several human diseases. In cancer, however, HSR activation has been associated with both anti- and pro-apoptotic responses. Heat-induced expression of cytoprotective and antiapoptotic heat shock proteins (HSP) is a known complication of hyperthermia, resulting in cancer cell thermotolerance and chemoresistance. In some instances, however, HSF1 activation may result in apoptosis induction. We have developed a library of novel potent inducers of HSF1 which are characterized by pro-apoptotic activity in several types of chemoresistant cancers. We have previously shown that HSF1 induction prevents TNFa- and mitogen-induced activation of NF-kB, a nuclear factor playing an important role in promoting inflammation, as well as cell proliferation and survival. NF-kB has been found to be constitutively activated in several types of chemoresistant cancers where it suppresses cell death pathways by switching on genes that dampen pro-apoptotic signals. We now show that activation of HSF1 by diverse chemical inducers, as well as by hyperthermia itself, results in inhibition of constitutive NF-kB activity and rapid down-regulation of the expression of NF-kB-dependent survival genes, triggering apoptosis in aggressive cancers presenting aberrant NF-kB regulation. The results suggest that the block of anti-apoptotic signaling pathways utilizing the IkB kinase IKK may play an important role in modulating HSR pro-apoptotic effects in chemoresistant cancers.

1C_03_S

Roles of HSF1 in inflammatory and immune response

Akira Nakai

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan, e-mail: anakai@yamaguchi-u.ac.jp

Inflammatory cytokines such as IL-1, IL-6, and TNF-a are induced in response to bacterial infection and diseases. These cytokines elicit the febrile response that is a complex physiological reaction to disease including cytokine-mediated rise in body temperature and activation of inflammatory systems. Fever plays beneficial roles on disease prognosis clinically. Experimentally, pretreatment of animals with heat shock also increases survival in a LPS-injected endotoxic model. These beneficial roles of fever are mediated partly by suppressing pyrogenic and inflammatory cytokines as expression of TNF-a, IL-1b, and IL-6 reduces in heat-shocked cells and in whole body exposed to high temperature. It was shown that HSF1 inhibits expression of cytokines by binding directly to TNF-a ?promoter, or by physically interacting with NF-IL6, an activator for IL-1b?. However, molecular mechanisms underlining fever-mediated suppression of cytokine gene expression are uncovered yet. Here we show that heat shock suppresses LPS-induced induction of IL-6 by activating HSF1 that induces ATF3, a negative regulator of IL-6. In vivo analysis using HSF1-null and ATF3-null mice reveals that HSF1 as well as ATF3 acts as a negative regulator of IL-6 expression in whole body and is required to inhibit fever. Unexpectedly, overexpression of ATF3 into cells has no effect on IL-6 expression in the absence of HSF1. HSF1 also binds directly to IL-6 promoter, and is required not only for a repressor ATF3, but also an activator NF-kB to bind to IL-6 promoter by partially opening chromatin structure. Taken together with the effects on expression of TNF-a and IL-1b, these results indicate that HSF1 plays a major role in feedback regulation of the febrile response.

1C_04_S

HSF2 influences the decision between proliferation and migration for neural cortical progenitors

Diane Trouillet, Anne Le Mouël, Rachid El Fatimy, Laurence Denis and Valérie Mezger

CNRS UMR8541, Ecole Normale Supérieure, 46 rue d’Ulm 75005 Paris

Heat Shock Factors (HSFs) are not only responsible for response to environmental stress, but are involved in developmental processes. We showed in collaboration with Lea Sistonen’s lab that HSF2 is involved in meiosis in both genders and in brain development (Kallio et al., 2002 ; Chang et al., 2006). We will describe how HSF2 influences the radial migration of young postmitotic neurons during cortical development, but also the proliferation of their neural progenitors (NPCs), using loss and gain of function models : Hsf2^-/- mice and the overexpression of HSF2 in the chick neural tube, by in ovo electroporation. The identification of new HSF2 target genes suggests us that HSF2 directly regulates genes that are involved in the control of microtubule dynamics, either in mitosis or during migration. The question is how HSF2, which is active in NPCs as well as in migrating postmitotic neurons, might differentially regulate distinct pools of target genes in these cell populations. The ability of HSF2 to differentially recruit transcription factors and chromatin modifiers as well as its distinct biochemical properties in NPCs versus postmitotic neurons has been investigated by biochemical and chromatin immunoprecipitation (ChIP) analyses and will be discussed. Such a combination of events and properties, coupled with the geography of HSF2 binding sites and of other transcription factors sites –characteristic of a given target gene – should allow HSF2 to induce or repress transcription and therefore to allow the decision for a NPC to continue to divide or to exit the cell cycle and start to migrate. Grant supports : ARC (Association pour la Recherche contre le Cancer) and ANR Neurosciences (Agence nationale pour la Recherche).

Developmental activity of HSF1 revealed by loss of function experiments

Elisabeth Christians

Traditional description of HSF1 activity is based on stress induction of molecular modifications which enable DNA binding and transcription of target genes. This view seems to imply that HSF1 is mainly inactive in normal, physiological situation.

Previously published data and the work presented here strongly suggest that this description needs to be revised.

Loss of function by gene targeting of Hsf1 in mice revealed a complex phenotype including HSF1 maternal requirement in the oocyte. Hsf1-/- females produce oocyte but they appear to be unable to properly accomplish expected developmental steps such as meiotic maturation, fertilization and activation, which are mandatory for embryonic development.

In order to better understand the link between HSF1 and those developmental steps, we searched for known and unknown targets of HSF1 in oocytes. Using RT combined with real time PCR, we show that Hsp genes are differentially expressed and regulated by HSF1 in oocytes under normal, physiological conditions.

Among HSF1 known targets, Hsp86 (Hsp90alpha), which is described as the inducible form of Hsp90, is significantly reduced in immature oocyte before meiotic maturation.

Additional experiments will be discussed to show how Hsp90 alpha can be one of the key link between HSF1 and early developmental steps undertaken by the oocytes.

Dual roles of Heat Shock Factor 1 (HSF1) in Cardioprotection, Pathologic Hypertrophy and Heart Failure in Transgenic Mice

András Orosz and Ivor Benjamin

andras.orosz@hsc.utah.edu

Heat shock factor 1 (HSF1), the major stress-inducible transactivator binds to heat shock elements (HSE) embedded in the promoter of heat shock proteins (HSPs) under stressful conditions and up-regulates the synthesis of HSP genes. In principle, HSF1 activation in the heart provides protection against ischemic insults and other stressful episodes through the increased synthesis and chaperone activities of HSPs. HSF1 level is also up-regulated in physiological cardiac hypertrophy following strenuous exercise regimens. To study HSF1 functions in the heart, we generated a transgenic mouse model expressing cardiac specific, inducible, but constitutively active HSF1 whose transcriptional competency does not require stressors. Two transgenic mouse lines were characterized, termed mHSF1(+) Low Tg and High Tg, with transgene expression ranging between equivalent and ~5-10 fold of the endogenous HSF1 level, respectively. In mHSF1(+) Low Tg mice, 7 days expression of mHSF1(+) induced ~3-fold upregulation of each major HSP groups, which conferred significantly increased ischemic protection in an ex vivo heart injury model. In contrast, doxycyclin feeding of mHSF1(+) High Tg line triggered ~5-10-fold increase in mHSF1(+) and target HSPs levels and ~30% cardiac hypertrophy in 7 days. Interestingly, 3 weeks mHSF1(+) expression resulted in decompensated cardiac hypertrophy, massive cell death and fibrosis along with greatly decreased cardiac function and overt heart failure in transgenic mice. Mechanistically, we hypothesize that upregulated class I and class II histon deacetylase family members, direct or indirect targets of mHSF1(+), may potentiate cardiomyocyte hypertrophy, whereas decreased expression of PGC-1a, a transcriptional co-activator of mitochondrial biogenesis accelerates the metabolic demise of mHSF1(+) High Tg hearts. Our studies have clearly established an intriguing dichotomy, based on low and high levels of HSF1 transactivation in both protective and pathological cardiac gene expression programs and, perhaps, human pathologies.

1D_01_S

HSP Release: Passive vs Active Release Mechanisms

Alexzander Asea

E-mail: asea@medicine.tamhsc.edu

Thus far, two mechanisms are recognized by which heat shock proteins (HSP) are released from cells into the extracellular milieu and subsequently into systemic circulation; a passive release mechanism as a result of necrotic cell death, severe blunt trauma, surgery and following infection with lytic viruses, and an active release mechanism which involves the non classical protein release pathway by which HSP70 is released as free HSP72 and within highly immunologically potent exosomes. This presentation covers the most recent findings on the mechanisms by which stress induces the release of HSP72 into the systemic circulation and addresses the biological significance of circulating HSP72 to host defense against disease.

1D_02_S

Hsp70 secretion and binding: its place in the immune response

Stuart K. Calderwood, Salamatu S Mambula, Jimmy Theriault and Jianlin Gong

Beth Israel Deaconess Medical center, Harvard Medical School and Center for Molecular Stress Response, Boston University Medical School.

Hsp70 plays a significant extracellular role within the immune response and can carry out both pro-inflammatory / pro-immune functions and anti-inflammatory effects depending on cellular and tissue context. However, understanding its place in the immune response requires deciphering the mechanisms by which it is released and the cell surface structures which it binds on target cells in the immune system.

We have examined mechanisms of Hsp70 release from tumor cells and macrophages and have found evidence for an active secretion mechanism. Our experiments indicate that hsp70 secretion employs a similar pathway to that used by other “leaderless” proteins such as interleukin 1b (IL-1b) involving the entry of Hsp70 into secretory lysosomes and ATP dependent release. Our evidence suggest however, that the trigger for hsp70 release is different from that which releases IL-1b: for instance, in macrophages exposed to E. coli, the trigger for IL-1b release is lipopolysaccharide, while Hsp70 release is triggered by a different signal. In addition, IL-1b release is independent of Hsp70 release and Hsp70 blocking does not alter release IL-1b.

Hsp70 released from cells binds to adjacent cells. Our experiments indicate that such receptors may include a diverse group of structures. Hsp70 signaling can be mediated by Toll Like receptors (TLR), CD40 and LRP/CD91. However, our recent studies indicate that Hsp70 internalization is mediated by scavenger receptors (SR) found on antigen presenting cells. We have found that at least 3 members of the SR including LOX-1, SREC-1 and FEEL-1/CLEVER-1 can bind and internalize Hsp70. As the SR play a profound role in the “cross presentation” of extracellular proteins to CD8+ T lymphocytes, these experiments suggest a pathway along which secreted Hsp70 with chaperoned peptide antigens can be taken up by APC and interact with immune cells.

1D_03_S

Extracellular heat shock proteins: searching for a role?

John H.H. Williams

Chester Centre for Stress Research, Department of Biological Sciences, University of Chester, Parkgate Road, Chester, CH1 4BJ, UK. E-mail: John.Williams@chester.ac.uk

Hsp70 can be detected in serum, despite being an intra-cellular protein. Hsp70 must therefore be released by damaged cells, or secreted by healthy cells. We have previously demonstrated that Hsp70 and Hsp60 are released from cells. An increasing number of cell types, including peripheral blood mononuclear cells (PBMCs), have been demonstrated to release Hsps, including Hsp70, Hsp60 and Hsp90.

This release of Hsp is stimulated, in vivo and in vitro, by a wide variety of stressors. Several cell types respond to extracellular Hsp exposure by releasing cytokines, such as TNF?. The type and source (human or bacterial) of Hsp alter the cytokine response. Extracellular Hsp70 protects cells from heat shock without needing to enter the cells. It is unclear whether the interaction of these extracellular hsps with the membrane is directly with lipid or protein.

The aims of this talk are to discuss:

• routes of Hsp release, and through this challenge the paradigm that only necrotic cells release Hsps.
• how extracellular Hsps interact with cells, and through this investigate their potential roles: as danger signals and as cell protectors.

1D_04_S

Quality Control of Extracellular Protein Folding: an Emerging Field

Justin J Yerbury, Amy R Wyatt, Stephen Poon and Mark R Wilson

School of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong. NSW. 2522. Australia.

Many serious human diseases are thought to arise from the effects of intra- or extracellular aggregates of proteins with non-native conformations. Inside cells, chaperone and protease systems regulate protein folding; however, little is currently known about any corresponding mechanisms that operate extracellularly. Knowledge of these mechanisms is important because it may lead to the development of new disease therapies. We have identified a small family of abundant human plasma proteins which have a potent small heat shock protein-like chaperone action. These proteins occur in plasma at levels of 0.1-2.0 mg/ml and thus in this locale are orders of magnitude more abundant than normally intracellular chaperones (e.g. Hsp70) found extracellularly¹. The abundant extracellular chaperones (ECs) form stable soluble complexes with misfolded and partially unfolded proteins to inhibit amorphous protein aggregation induced by physical or chemical stresses. The ECs also exert potent effects on amyloid formation and toxicity². Recent data suggests that complexes formed between ECs and "damaged" (misfolded/unfolded) proteins are rapidly bound by cell surface receptors in vivo and disposed of by receptor-mediated endocytosis and lysosomal degradation. Thus, a model is emerging in which ECs patrol extracellular spaces, bind to damaged proteins when present and mediate their rapid clearance and disposal. Disease pathologies associated with protein aggregation may arise when this system is overloaded. 

1D_05_S

Extracellular Hsp72: A Double-Edged Sword for Health

Monika Fleshner

Environmental or emotional challenge triggers a cascading series of physiological responses which are collectively termed the “stress response”. The stress response can be assessed at the behavioral, neural, hormonal, immunological and single cell, levels and evolved to benefit an organism’s chance of survival during times of acute challenge. The stress response has been studied for many years, however, its impact on specifically immune function has only recently been appreciated. Acute activation of the stress response has both inhibitory and stimulatory effects on immunity. The focus of this presentation is on a novel mechanism for the immunostimulatory effects of stress.  Specifically, we propose that an endogenous, ubiquitous cellular stress protein, heat shock protein 72, when found in the extracellular environment may contribute to stress-induced potentiation of innate immunity. We develop the hypothesis that the release of extracellular heat shock protein 72 (eHsp72) is a normal feature of the acute stress response that can have either positive or negative consequences for host defense depending on several factors, including the nature of the eHsp72 (naked versus antigen-associated), and host health status (absence or presence of pre-existing inflammatory disease). Thus, stress-induced eHsp72 release may be a double-edged sword for host defense.

1D_06_S

Hsp70 is secreted from cells in vesicles that resemble lipid rafts

A. De Maio^{1, 2}, M. Rodríguez¹, T. Frey², M. Gehrman³, D. Nino¹, J.C. Diaz ⁴, C. Steinem⁵, N. Arispe⁴, G. Multoff³, and V. Vega¹

1Department of Surgery, University of California San Diego, La Jolla, California, USA; ²Johns Hopkins University, Baltimore, Maryland, USA; ³Klinikum Rechts der Isar, Technical University, Munich, Germany;⁴Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA;⁵Institute for Organic and Biomolecular Chemistry, University of Goettingen, Göttingen, Germany

Heat shock proteins (hsp) play a major role in a variety of cellular processes during normal conditions as well as during the restoration of homeostasis after stress. Recently, Hsp70, the major stress-induced hsp, has been found in the extracellular medium. Moreover, Hsp70 has been observed to activate cells of the immune system. Thus, circulating Hsp70 is thought to act as a danger signal that triggers the response to injury. We have previously reported that Hsp70 is capable of interacting with lipid membranes and has a high selectivity for phosphatidylserine (PS). The incorporation of Hsp70 within PS-containing membranes was enhanced by the presence of spingolipids (GM1) and cholesterol, which are major components of lipid rafts. In fact, Hsp70 was detected in the lipid raft fraction isolated from Triton X-100 solubilized HepG2 cells after heat shock (HS). The presence of Hsp70 in this fraction was reduced by depletion of cellular cholesterol by treatment with ß-cyclodextrin. Hsp70 was visualized within the plasma membrane of HepG2 cells after HS, co-localizing with lipid raft markers. Analysis of the extracellular medium of HepG2 cells after heat shock revealed the presence of Hsp70 within membranes that can be isolated by high speed centrifugation. These membranes containing Hsp70 were rich in GM1 and cholesterol, but depleted of other cellular components, such as actin. Moreover, the Hsp70 within these extracellular membranes was resistant to Triton X-100 solubilization. Thus, extracellular membranes containing Hsp70 resemble lipid rafts. We propose that part of the extracellular Hsp70, which may be involved in the activation of immune cells, is present in the membrane derived from lipid rafts.

Module 1 – Poster lectures:

1A_01_P

HspB8 and Bag3: a new chaperone complex stimulating mutated huntingtin degradation by macroautophagy

Carra S * (1), Seguin S (1), Vinet J (2), Lambert H (1), Sik A (2) and Jacques Landry (1)

(2) Centre de recherche Robert-Giffard, 2601 Chemin de la Canardiere, Québec, Canada

HspB8 is a member of the small heat shock proteins or HspB family of molecular chaperones, which comprises ten members in mammals (HspB1-10). We previously demonstrated that, in cultured cells, overexpression of HspB8, but not of HspB1 or HspB5, totally blocked the insolubilization and accumulation of a pathogenic aggregation-prone form of Huntingtin (Htt43Q). Here we report that HspB8 stably and sotichiometrically interacts with the co-chaperone Bag3. HspB8 association with Bag3 is essential for both its structural stability and chaperone function, as demonstrated by knocking down Bag3 expression by siRNA technique. We next investigated the chaperone function of the HspB8/Bag3 complex. We found that Bag3 overexpression, like HspB8, facilitated Htt43Q degradation in cultured cells. Treatment with specific macroautophagy inhibitors dramatically decreased the HspB8/Bag3 chaperone activity and lead to the accumulation of aggregated Htt43Q. Moreover, HspB8 and Bag3 both increased the number of cells containing LC3 positive-vacuoles and stimulated the lipidation of LC3, a step which is necessary for LC3 incorporation into the autophagosomes. These results strongly suggest the implication of the macroautophagy process in the HspB8/Bag3 complex mechanism of action. By joining the ability of recognizing endogenous misfolded proteins and of stimulating the macroautophagic vacuole formation, the new HspB8/Bag3 chaperone complex may play a crucial role in the protein quality control system responsible for eliminating potentially harmful aggregating proteins.

1A_02_P

1A_03_P

New Ideas on Mechanism of Protein Aggregation and Mechanism of Protective Action of a -Crystallin

K.A. Markossian and B.I. Kurganov

Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Leninsky pr. 33 e-mail: markossian@inbi.ras.ru, boris@kurganov.com

The kinetics of thermal aggregation of proteins (b -crystallin from calf eye lens, glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle and mitochondrial aspartate aminotransferase from porcine heart) were studied by dynamic light scattering. On the basis of the study of aggregation kinetics a new mechanism of protein aggregation was developed. The first stage of protein aggregation is the stage of the formation of the start aggregates. The size of the start aggregates was estimated by construction of the light scattering intensity versus hydrodynamic radius plots. The hydrodynamic radius of the start aggregates remains constant at variation of temperature and the protein concentration. The second stage of the aggregation process is sticking together of the start aggregates. This stage proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the particles (the regime of “diffusion-limited cluster-cluster aggregation”). Under this regime each collision of the interacting particles results in their sticking together. Suppression of thermal aggregation of proteins in the presence of a -crystallin is due to diminishing of the size of the start aggregates, increase in the duration of the latent period over which the start aggregates are formed, and transition of the aggregation process into the kinetic regime wherein the sticking probability for the colliding particles becomes less than unity (the regime of “reaction-limited cluster-cluster aggregation”).

The study was funded by the Russian Foundation for Basic Research (grants 05-04-48691-a and 06-04-39008-a), Program “Molecular and Cell Biology” of the Presidium of the Russian Academy of Sciences.

1A_04_P

Transduced Tat-Hsp40 protects cells against oxidative stress

Jung-Hoon Yoon¹, Soo-A Kim² and Sang-Gun Ahn^1,*

* corresponding author

Abstract

Heat shock protein (Hsp) 40 acts as a co-chaperone of Hsp70 by facilitating the ATPase activity of Hsp70 as well as promoting the protein folding and renaturation by Hsp70. In the present study, Hsp40 gene was fused with a gene fragment encoding the HIV-1 TAT protein transduction domain (YGRKKRRQRRR) in a bacterial expression vector pTAT-HA to produce the TAT-fused Hsp40 (TAT-Hsp40). Purified TAT-Hsp40 was effectively transduced into the HEK 293 cells in a time- and dose-dependent manner. To examine the effect of TAT-Hsp40 upon oxidative stress, HEK293 cells were exposed to H₂O₂. Oxidative stress induced the rapid increase of proteasome activity followed by cell death in HEK 293 cells. However, HEK 293 cells transduced by TAT-Hsp40 showed resistance against oxidative stress-induced cytotoxicity. TAT-Hsp40 transduced cells showed decreased proteasome activity and inhibited Hsp70 degradation. These results suggest that Hsp40 might protect cell death from oxidative stress by preserving the cellular level of Hsp70.

Keywords: Hsp40, Hsp70, TAT, proteasome, oxidative stress 

1A_05_P

Effect of alpha-crystallin on thermal denaturation and aggregation of mitochondrial aspartate aminotransferase

N.V. Golub, K.A. Markossian

Bach Institute of Biochemistry RAS, 33 Leninsky av., Moscow, 119071, Russia (ngolub@inbi.ras.ru)

Thermal denaturation and aggregation of mitochondrial aspartate aminotransferase have been investigated by differential scanning calorimetry (DSC) and dynamic light scattering. The dependence of excess heat capacity of AAT on temperature passes through a maximum at 72.4 degrees C and can be described by a scheme in which the denaturation process is considered as an irreversible first-order reaction. Inactivation of AAT follows the exponential law. The parameters of Arrhenius equation have been determined from the DSC data and temperature dependence of the inactivation rate constant. Calculations show that at 57.5 degrees C the inactivation constant (k_in) is 28.9 times higher than the denaturation rate constant (k_den) determined from the DSC data. The k_in/k_den ratio decreases with temperature and becomes equal to 1.3 at 77 degrees ?. It has been shown that alpha-crystallin, the protein possessing a chaperone-like activity, reduces thermostability of ??? and accelerates thermoinactivation of the enzyme. Suppression of thermal aggregation of ??? in the presence of alpha-crystallin has been demonstrated. The protective effect of alpha-crystallin is due to the decrease in the size of the start aggregates and transition of the aggregation process from the regime of diffusion-limited cluster-cluster aggregation (the sticking probability for the colliding particles is equal to unity) to the regime of reaction-limited cluster-cluster aggregation (the sticking probability for the colliding particles is less than unity). The study was supported by the Russian Foundation for Basic Research (grants 05-04-48691-a and 06-04-39008), Program “Molecular and Cell Biology” of the Presidium of the Russian Academy of Sciences and INTAS (grant 03-51-4813).

1A_06_P

HSP26 from Saccharomyces cerevisiae is a polydisperse small heat-shock protein

JA Aquilina, JLP Benesch, RA Lindner, A Rekas, E Vierling, CV Robinson, JA Carver

University of Wollongong, Northfields Avenue, Wollongong, NSW, Australia 2522, email: aquilina@uow.edu.au

Here we report investigations into the oligomeric organization and dynamics of the Saccharomyces cerevisiae small heat-shock protein ScHSP26. Using both multi-angle light scattering and mass spectrometry (MS) approaches we have shown that at ambient temperature this protein is exists as a polydisperse assembly, comprising numerous oligomeric states. Relative quantification of these oligomeric states using a tandem MS technique demonstrated the 24mer to be anomalously abundant, consistent with previous reports which have culminated in a 3D structure for this oligomeric state. Significantly, we demonstrate for the first time, that ScHSP26 also forms larger oligomers, up to 40 subunits in size at room temperature. The oligomers observed were exclusively composed of an even number of subunits. This strongly suggests there to be a basic dimeric ‘building block’, an observation consistent with the 3D structure of the ScHSP26 24mer, as well as studies performed on sHSPs from bacteria, plants and mammals. Using an online thermo-controlled device we investigated the effect of heat stress on the oligomeric structure of ScHSP26. We found that as the temperature was increased, dissociation of the oligomers into dimers and monomers was observed. Such dissociation has previously been observed for ScHSP26 and other sHSPs. The remaining ScHSP26 existed in forms 32 subunits or larger, with the majority having assembled into a 40mer. There is a possibility that these larger forms are unusually resistant to thermal stress, or ScHSP26 preferentially associates into these forms at elevated temperatures.

1A_07_P

Small Hsps form Intracellular Granules with Akt/PKB and p38 distinct from TIA/TIAR-containing Stress Granules

Anastassiia Vertii, Alexey Kotlyarov, and Matthias Gaestel

From the Institute of Biochemistry, Medical School Hannover, Hannover 30625, Germany

Small heat shock proteins are rapidly phosphorylated in response to cellular stress upon activation of p38 kinase pathway. Phosphorylation occurs at Ser15 and Ser86 in mouse Hsp25 and at Ser15, Ser78 and Ser82 in human Hsp27 resulting in changes in their oligomeric state. Stress causes accumulation of Hsp25/27 in an insoluble fraction and formation of intracellular granular structures. Although stress-induced Hsp27 granules have been known for a long time, the presence of other proteins in these granules remains enigmatic. Certain stress granules (SGs) contain mRNA-binding proteins, such as TIA/TIAR, and sequested processing bodies with mRNA and translation initiation factors which play a role in translational inhibition. Here we report stress-independent interaction between Hsp25 and Akt/PKB and characterised the regions in Hsp25 and Akt responsible for this interaction. We further analysed stress-induced insolubilisation of small Hsps and demonstrate stress-induced insolubilization of Akt together with Hsp25. By immunofluorescence we show that Hsp25/27-containig SGs, which are formed in response to arsenite treatment of HeLa cells, include Akt and p38. Although it is known that MK2 interacts with p38 we were not able to detect overexpressed MK2 in these SGs. Furthermore, these SGs are distinct from cytoplasmic granules that contain TIA/TIAR. siRNA silencing and nuclear/cytoplasmic fractionation was used to further describe the role of these novel SGs for the function of Akt.

1A_08_P

Small heat shock proteins effectively protect myosin S1 and F-actin from thermally induced aggregation

A. V. Pivovarova³, D. I. Markov², N. B. Gusev² and D. I. Levitsky¹

¹A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow,
²Department of Biochemistry, School of Biology,
³ School of Bioengeneering and Bioinformatics, Moscow State University, Moscow, Russia

We have found that small heat shock proteins (sHsp) do not affect thermal denaturation of actin filaments (F-actin) measured by differential scanning calorimetry (DSC), but effectively prevent their heat induced aggregation. The presence of sHsp significantly increases the temperature of F-actin aggregation. It has been shown by co-sedimentation experiments that sHsp do not bind to native F-actin. However, after heating to 75oC they form soluble complexes with thermally denatured actin, which do not precipitate at high-speed centrifugation, unlike F-actin in the absence of sHsp that fully precipitates under the same conditions. Sedimentation coefficients of these complexes, as estimated by analytical centrifugation, and their size (as determined by dynamic light scattering experiments) were much less than these parameters for native F-actin and thermally denatured actin. The gel filtration was used to estimate a stoichiometry sHsp/actin in these complexes. We also studied the effects of sHsp on thermal denaturation and aggregation of isolated myosin head (myosin subfragment 1, S1). It has been shown that under heat shock conditions (upon incubation at 43oC) sHsp effectively prevent aggregation of S1 induced by its thermal denaturation, but these proteins have no appreciable influence on the thermal unfolding of S1, as studied by DSC, and on thermally induced inhibition of myosin ATPase. Supported by grants from RFBR and the Program "Molecular and Cellular Biology" of Russian Academy of Sciences.