It has become clear that all types of cells contain a wealth of small, non-coding RNAs (sRNAs) that have important roles in regulating gene expression at the post-transcriptional level. In the bacterium Escherichia coli genome-wide searches have led to the identification of ~ 100 sRNAs and many of these are known or believed to act by base-pairing to modify the translation and/or the stability of target mRNAs. Generally, the regulation relies on short stretches of base pairing, which allows recognition of multiple target mRNAs by a single sRNA as well as the recognition of a single target by multiple sRNAs. Regulatory RNA molecules acting in this manner require the RNA chaperone Hfq, which promotes the pairing of complementary RNA molecules. Another feature of the chromosomally encoded sRNAs is that they are tightly regulated at the transcriptional level, and frequently expressed as part of global regulatory networks which function in response to environmental stress signals. - Here, I will focus on the identification of two highly conserved ?E-dependent sRNAs, RybB and MicA, and discuss recent progress in our understanding of the ?E -signalling system, which monitors the bacterial cell envelope. Studies in E. coli and Salmonella demonstrate that MicA and RybB act within the envelope stress response and serve dual roles. First, when inducing stress occurs, they facilitate rapid shut-off of the synthesis of multiple outer membrane proteins. Moreover, in unstressed cells, the two sRNAs function within a surveillance loop to maintain envelope homeostasis and to achieve autoregulation.

Johansen J et al.: Conserved small non-coding RNAs that belong to the s E regulon: role in down-regulation of outer membrane proteins. J Mol Biol 2006, 364: 1-8. Papenfort et al.: s E-dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA decay. Molecular Microbiology 2006, 62: 1674-1688. Valentin-Hansen et.al.: Small RNAs controlling outer membrane porins. Current Opinion in Microbiology 2007, in press

2C_02_S

Stress-induced nuclear bodies and transcription of repeated sequences

Caroline Jolly, Angéline Eymery, Sabrina Fritah, and Claire Vourc’h

Corresponding author : caroline.jolly@ujf-grenoble.fr

Exposure of cells to stress induces dramatic changes in gene expression, activating the expression of certain genes such as those encoding the heat shock proteins or HSPs, and inactivating others. In paralell to the activation of hsp gene expression, we have shown that heat shock also induces the formation of particular nuclear structures termed nuclear stress bodies or nSBs. These structures form principally on the pericentromeric region of human chromosome 9 (9q12) through a direct binding of HSF1 with satellite III repeated sequences. We have shown that heat shock induces the transcription of these repeated sequences into non-coding RNAs termed satellite III transcripts. This transcription is RNA-polymerase II- and HSF1-dependent. The function of the satellite III transcripts is still unknown, but several hypotheses can be considered. Since they remain associated with chromosome 9 for several hours after synthesis, they may play a role in chromatin structure. Alternatively, since several splicing factors remain associated to the sat III transcripts, they could play a role in the regulation of alternative splicing, a function which is indeed altered during heat exposure. I will present our latest findings concerning satellite III transcripts and discuss their possible function during stress exposure.

2C_03_S

Eukaryotic RNA Thermosensor

Ilya Shamovsky, June Hyung Lee, Maria Vera, Konstantin Shatalin, and Evgeny Nudler

Department of Biochemistry, New York University School of Medicine, New York, NY, 10016, USA

The heat shock transcription factor (HSF1) plays a central role in the heat shock (HS) response in eukaryotes by inducing the expression of heat shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed mammalian cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. We have shown that HSF1 activation by HS is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a novel non-coding RNA, which we termed HSR1 (Heat Shock RNA-1). Both HSR1 and eEF1A are required for HSF1 activation in vitro. Antisense oligonucleotides or siRNA against HSR1 impair the HS response in vivo, rendering mammalian cells thermosensitive. We also show that non-coding RNAs homologous to mammalian HSR1 are present in other eukaryotic species including Xenopus, Drosophila and C.elegans. HSR1 is constitutively expressed in all these organisms and its homologues are functionally interchangeable. Our results suggest a general model for eukaryotic HS genes activation whereby HSR1 serves as a cellular thermosensor that determines the temperature threshold for the HS response; while eEF1A links HSP expression to major cellular perturbations during HS, such as translational shutdown and cytoskeleton collapse. The central role of HSR1 during HS implies that targeting this RNA could serve as a new therapeutic mode for cancer, inflammation and other conditions associated with HSF1 deregulation.

2C_04_S

Non-coding hsrw RNA and post-transcriptional processing in stressed cells

Moushami Mallik and Subhash C. Lakhotia

Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India ( lakhotia@bhu.ac.in )

Every cell needs a large variety and number of proteins for transcription and processing of nascent transcripts (splicing, other RNA processing, transport etc). Stress or non-permissive conditions, which largely inhibit transcriptional and RNA processing machineries, result in high surplus of unengaged RNA processing proteins. Since these proteins are not degraded but must be available upon recovery, they need to be reversibly sequestered. Heat stress induced formation of nuclear stress bodies/granules in human cells and clustering of the varieties of nuclear speckles (IGCs, paraspeckles, omega speckles etc) in different cell types appear to reflect such sequestration. A major focus of studies in our laboratory is on the developmentally expressed and stress-inducible non-coding hsrw gene in Drosophila. The large nuclear transcript of this gene, hsrw-n, is known to be required for organizing the unengaged nuclear hnRNPs and related RNA-binding proteins in nucleoplasmic omega speckles in nearly every cell type of Drosophila. Using transgenic lines designed to either over-express or ablate the hsrw-n transcripts, we reconfirm that the large nuclear hsrw-n transcript is required for formation of omega speckles and show that this non-coding transcript plays crucial roles in normal development and is also essential for survival of the organism following stress. We believe that such non-coding RNA species perform a wide range of functions in cells through their ability to interact with a great variety of proteins and thus act as “hubs” to integrate complex networks of gene activity during development and under conditions of stress.

2D_01_S

Stress in-and-out

Gábor Bánhegyi

Department of Medical Chemistry, Semmelweis University, 1444 Budapest, POB 260, Hungary. E-mail: banhegyi@puskin.sote.hu

Glucocorticoids are main actors in the pathomechanism of stress. In the original context formulated by Hans Selye in his stress theory, glucocorticoids are produced in the adrenal cortex upon the activation of the hypothalamic - pituitary - adrenal axis. Their increased production mediates alarm reactions in acute stress, facilitating metabolic alterations in the general adaptation syndrome allowing the individual to attempt countermeasures such as the “fight or flight” response. Recent observations show that active glucocorticoids can be also formed from their inactive counterparts in various tissues, including liver and adipose tissue. This prereceptorial activation takes place in the lumen of the endoplasmic reticulum (ER) and depends on the activity of the glucose-6-phosphate transporter - hexose-6-phosphate dehydrogenase - 11ß-hydroxysteroid dehydrogenase type 1 triad. Increased prereceptorial glucocorticoid activation is accompanied by the signs of ER stress in certain human diseases (obesity, metabolic syndrome, type 2 diabetes). These diseases are more common among socio-economically disadvantaged individuals and are associated with lifestyle factors and chronic stress. It has been recently suggested that the ER can function as a sensor for electron donors and acceptors, i.e. nutrients and oxygen. In the current social environment of high energy input and minimal physical activity, the ER encounters a nutrient (electron) overload, leading to a redox imbalance in the lumen, which is the most frequent cause of ER stress and consequent apoptosis. Furthermore, reductive effects favor the increased prereceptorial glucocorticoid activation. In conclusion, glucocorticoid response can be initiated by an autonomous sensing of (nutrient) stress in cellular level beside the central neuroendocrine mechanism.

2D_02_S

ER Stress Induction of UPR Regulator GRP78: Role in Development and Disease

Amy S. Lee, Peter Baumeister, Changhui Mao, Miao Wang, Risheng Ye, Min Ni, Yong Fu, Jianze Li and Dezheng Dong

Department of Biochemistry and Molecular Biology and the USC/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, 1441 Eastlake Ave., Los Angeles, CA 90089-9176. E-mail: amylee@usc.edu

Mammalian cells have evolved multiple adaptive pathways, referred to as the unfolded protein response (UPR), that allow them to respond to perturbations in endoplasmic reticulum (ER) homeostasis. One major pro-survival mechanism is mediated by the ER chaperone GRP78/BiP, an anti-apoptotic protein which also regulates ER stress signaling. To probe the physiologic function of GRP78/BiP, mouse models were recreated targeting the Grp78 allele. This lead to the discovery that complete depletion of GRP78 results in early embryonic lethality due to proliferation defects and apoptosis of the inner cell mass which is the precursor of embryonic stem cells. Our results show that reduction of GRP78 level by half is sufficient to maintain cellular homeostasis during development with no major consequence in ER stress signaling. This implies that an elevated GRP78 level is more critically needed in cells undergoing physiological or pathological stress, as exemplified by protection of vulnerable neuronal cells and allowing cancer cells to evade the host defense system and cancer therapies. In this lecture, we will discuss the consequence of conditional knockout of GRP78 in specific neuronal cells. Due to hypoxic conditions and glucose deprivation caused by poor vascularization, the microenvironment of tumors represents physiological ER stress and the UPR is activated for tumor cell survival. In this lecture, we will discuss how GRP78 deficiency will affect cancer progression and the underlying mechanisms responsible for the phenomenon.

Ischemia/Stroke Impairs Endoplasmic Reticulum Function

Wulf Paschen, Ph.D.

Multidisciplinary Neuroprotection Laboratories, Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina, USA, wulf.paschen@duke.edu

2D_04_S

Managing and exploiting stress in the antibody factory

Roberto Sitia

Universita Vita-Salute San Raffaele Scientific Institute, 20132 Milano, Italy, Fax +39 02 2643 4723, E-mail: r.sitia@hsr.it

Upon encounter with antigen, long-lived B lymphocytes differentiate into short-lived plasma cells, the terminal effectors of the humoral immune response. Plasma cells are specialized in immunoglobulin (Ig) secretion, each of them being capable of releasing thousands molecules per second. How do plasma cells achieve such an efficiency? How do they cope with metabolic and redox imbalances that exuberant protein secretion can cause? Is plasma cell death linked to Ig production, such as to limit antibody responses? We have dissected terminal plasma cell differentiation through dynamic imaging, proteomics and genomic analyses. Our results show that waves of functionally related proteins are produced to increase the capacity of the antibody factory, and shed some light in the signalling pathways utilised to orchestrate massive de novo ER biogenesis. As to the mechanisms that lead to plasma cell death, we showed that in the late phases of plasmacytic differentiation, when antibody production becomes maximal, proteasomal activity unexpectedly decreases. The excessive load for the reduced proteolytic capacity correlates with accumulation of polyubiquitinated proteins, stabilization of endogenous proteasomal substrates (including Xbp1s, Ik-Ba and Bax), onset of apoptosis, and sensitization to proteasome inhibitors. A developmental program seems therefore to link plasma cell death to protein production, explaining the peculiar sensitivity of normal and malignant plasma cells to proteasome inhibitors.

2D_05_S

Endoplasmatic reticulum and energy stress response mechanisms in intestinal epithelial cells under chronic inflammation: inhibitory effects of Interleukin 10

Messlik A., Ruiz P. A., Kim S. C., Sartor R. B., Haller D.*

email: haller@wzw.tum.de; phone: ++49-(0)8161-712026; fax: ++49- (0)8161-712097

The initiation of endoplasmic reticulum (ER) stress responses and energy deficiency in intestinal epithelial cells (IEC) may contribute to the pathogenesis of chronic intestinal inflammation. The aim of this study was to characterize anti-inflammatory mechanisms of interleukin 10 (IL10) using functional epithelial cell proteomics.

Proteome analysis from primary IEC of Enterococcus faecalis- and Escherichia coli monoassociated IL10 deficient (IL10-/-) mice revealed increased expression levels of the glucose-regulated ER stress proteins (grp)78 under conditions of experimental colitis. Interestingly, the induction of ER stress response mechanisms under conditions of chronic inflammation was associated with decreased expression levels of the mitochondrial creatine kinase and increased activation of the AMP kinase system in primary IEC, suggesting dysregulation of the cellular energy homeostasis. Most importantly, IL10 inhibited grp78 expression in IL10 receptor reconstituted epithelial cells. Chromatin immunoprecipitation analysis revealed that IL10-mediated p38 signaling inhibited TNF-induced recruitment of the ER-derived activating transcription factor (ATF)-6 to the grp78 promotor likely through the blockade of ATF-6 nuclear translocation.

The failure of energy homeostasis in primay IEC from inflamed IL10-/- mice was associated ER stress responses in the intestinal epithelium. In addition, IL10 inhibits inflammation-induced ER stress response mechanisms by modulating ATF-6 nuclear recruitment to the grp78 gene promotor.

2D_06_S

Proteasomal degradation is transiently arrested during inhibition of translation in ER stress

Marina Shenkman and Gerardo Z. Lederkremer

The unfolded protein response (UPR) activates transcription of genes involved in proteasomal degradation. However, we found that in its early stages the UPR leads to a transient inhibition of proteasomal disposal of cytosolic substrates (p53 and p27Kip1) and of those targeted to ER-associated degradation (uncleaved precursor of asialoglycoprotein receptor H2a). Degradation resumed soon after the protein synthesis arrest that occurs in early UPR subsided. Consistently, also protein synthesis inhibitors blocked ubiquitin/proteasomal degradation. Ubiquitination was inhibited during the translation block, suggesting short-lived E3 ubiquitin ligases as candidate depleted proteins. This was indeed the case for p53 whose E3 ligase, MDM2, when overexpressed,restored the degradation, whereas a mutant MDM2 in its acidic domain restored the ubiquitination but not completely the degradation. Inhibition of proteasomal degradation early in UPR may prevent depletion of essential short-lived factors during the translation arrest. Stabilization of p27 through this mechanism may explain the cell cycle arrest in G1 when translation is blocked by inhibitors or by the UPR.

2E_01_S

Gas sensing in the nervous system: hypoxia and potassium channels

P. J. Kemp, S. P. Brazier, N. Baban, D. Riccardi, C. T. Müller, S. J. Williams

School of Biosciences, Cardiff University, U.K. Kemp@cf.ac.uk.

Whether part of the normal intra-uterine development, at high altitude or in pathological conditions, hypoxia is one of the most common stresses to which an organism may be exposed and the ability to adapt to such changes in blood gases is crucial for optimal delivery of molecular oxygen to respiring tissues. The principal sensory component of this homeostatic mechanism is the carotid body. Ideally situated in the bifurcation of the common carotid artery, they respond muliplicatively to hypoxia, hypercapnia, pH and hypoglycaemia. At the cellular level, hypoxia promotes inhibition of plasma membrane the Ca²⁺-activated, K⁺ channel (BK_Ca) of carotid body glomus cells which leads to Ca²⁺ influx and transmitter release. Functional proteomics has recently demonstrated that hemeoxygenase-2 (HO-2) is an O₂ sensor linking hypoxia to BK_Ca inhibition (Williams et al., 2004 Science 306, 2093-2097), 2004; a process which depends upon carbon monoxide (CO) as the second messenger. The mechanism of such gas/channel interactions is complex, and may involve interactions with heme (Jaggar et al. Circ Res 97, 805-812, 2005. Using chemical and molecular modifications of the BK_Ca ?-subunit in combination with channel chimera studies, we are now beginning to appreciate the kinetic and structural basis of the dynamic regulation of BK_Ca by endogenous production of CO. Taken together, we have proposed a model of how HO-2 functions as a sensor of acute reductions in environmental O₂. Thus, in normoxia, the protein partnership of HO-2 and BK_Ca optimizes the permissive effect of CO. However, during the stress of hypoxia, the balance between intracellular heme concentration and the evolution of cellular CO is altered, thereby promoting channel inhibition and activation of the carotid body.

2E_02_S

Oxygen, a source of Life and Stress – When Hypoxia meets Cancer

E. Berra, M. C. Brahimi-Horn, J. Chiche, F. Dayan, R. Garcia-Medina, A. Ginouves, N. Mazure, D. Roux, M-P. Simon and J. Pouysségur

Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice, France. pouysseg@unice.fr

The development of an oxygen-rich atmosphere has been one of the most important events in the history of life on Earth. Oxygen with its exceptional reactivity, represents the far most potent natural pollution on our planet, promoting Life or Death. About 2.4 billion years ago the high amount of dissolved and free oxygen, produced by photosynthesis, in the oceans and atmosphere has driven to extinction most anaerobic organisms. Over the past 500 million years, oxygen levels fluctuated between 15 and 35% imposing constant stress on and subsequent stringent evolution of living organisms.

During mammalian embryonic development or in the context of tumor expansion, proliferating cells rapidly outstrip the supply of nutrients. Although cells sense and respond to variations in concentrations of all nutrients, oxygen sensing has emerged as a central control mechanism of vasculogenesis. Whereas a decrease in the pO₂ (hypoxic stress) induces angiogenesis, an increase in pO₂ (hyperoxic stress) induces vascular pruning. Oxygen concentrations ‘sculpt’ the blood vascular network of vertebrates. At the heart of this regulatory system is the Hypoxia-Inducible Factor, HIF, which interestingly controls, among other gene products, the expression of VEGF-A and Angiopoietin-2 (Ang-2), two key angiogenic factors. This finding has therefore placed the hypoxia-signaling pathway at the forefront of nutritional control. Rapidly activated upon a hypoxic stress, HIF induces a vast array of gene products inducing cell-, tissue-, and organismal-survival. Among the HIF-controlled functions are inhibition of ATP-consuming processes (protein and lipid synthesis), inhibition of mitochondrial respiration (to save O₂), increase in anaerobic glucose metabolism, regulation of intracellular pH, increased angiogenesis and cell migration, and so HIF has become recognized as a strong promoter of tumor growth. This pro-oncogenic feature is only one facet of the dual action of HIF. Besides being a ‘guardian’ of oxygen homeostasis, HIF is capable of inducing pro-apoptotic gene products (BNIP3, BNIP3L) that are in fact pro-survival by inducing autophagy. The molecular mechanism leading to this survival process that is strictly controlled by a drop in pO₂ will be presented.

2E_03_S

Hypoxia and Inflammation

Cormac T. Taylor

UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. cormac.taylor@ucd.ie

The chemical reduction of molecular oxygen which occurs during mitochondrial oxidative phosphorylation is the main source of metabolic energy for virtually all eukaryotic cells. Decreased tissue oxygen supply (leading to hypoxic stress) is a common feature in a range of disease states where inflammation occurs including inflammatory bowel disease (IBD), arthritis, cancer, atherosclerosis and stroke. Recent studies indicate that hypoxia promotes inflammatory signaling pathways through specific mechanisms involving altered hydroxylation of specific residues on key transcription factors including (but likely not limited to) the hypoxia inducible factor (HIF-1) and nuclear factor kappa B (NF-kB). Thus it appears that hypoxia plays an important modulatory role in inflammatory disease development. It appears that a family of prolyl- and asparaginyl-hydroxylases are key common oxygen sensors in conferring hypoxic sensitivity to these pathways. These hydroxylases are absolutely dependent upon the presence of molecular oxygen for activity and are thus inhibited in hypoxia leading to derepression of transcriptional effectors. A greater understanding of the oxygen sensing and signaling mechanisms leading to the activation of these transcriptional responses to hypoxia will allow the development of novel therapeutics in a range of disease states where hypoxia and inflammation are co-incidental events.

2E_04_S

Stress regulated bHLH/PAS transcription factors: the Dioxin Receptor and Hypoxia Inducible Factors

M.L Whitelaw, S. Linke, A. Chapman-Smith, T. Wallis, K. Dave, J. Gorman and D.J. Peet

murray.whitelaw@adelaide.edu.au

The basic Helix-Loop-Helix / Per-Arnt-Sim (bHLH/PAS) family of transcription factors perform essential functions during early development and help maintain homeostasis in the adult. For example, the Hypoxia Inducible Factors (HIF-1a and HIF-2a proteins) play a major role in angiogenesis and cellular adaption to low oxygen stress. At normoxia, two oxygen dependent hydroxylases posttranslationally modify specific proline and asparagine residues of the HIFs, severely dampening their activity. During hypoxia, the HIFs exhibit dramatic increases in both protein stability and intrinsic transactivation capacity, due to attenuation of these two hydroxylases. The Dioxin Receptor (or Aryl hydrocarbon receptor) responds to the stress of xenobiotic infiltration by inducing a battery of genes for xenobiotic metabolism. The Dioxin Receptor (DR) is also the mediator of toxic responses to dioxins and PCBs. Both HIFs and the DR need to heterodimerise with a central bHLH/PAS partner protein, termed Arnt, to form active transcription factor complexes. The PAS domain provides a critical protein interaction surface during dimerisation and in the case of the DR, functions as a signal regulated domain. During our studies of stress induced activation mechanisms of the HIFs and DR, we have found recurring themes of posttranslational modification and an important role of the PAS domain in allowing the bHLH to bind to non-canonical E-box sequences of DNA. The presentation will present data to illustrate and expand upon these themes.

2E_05_S

Specific FKBP38 inhibitor reduces hypoxia-induced apoptosis in ventricular myocytes from adult and neonatal rat hearts

S. Rau², B. Husse¹, D. Wildemann², M. Gekle¹, G. Fischer²

britta.husse@medizin.uni-halle.de

Hypoxia/reoxygenation causes cell death of cardiomyocytes by a mitochondrion-dependent pathway. The Ca²⁺/CaM activated FK506-binding protein 38 (FKBP38) can interact with Bcl-2 through its PPIase active site and participates in the promotion of apoptosis. This study investigated the effect of specific FKBP38 inhibition with DM-CHX (N-(N',N'-dimethylcarboxamidomethyl)-cycloheximide) on the hypoxia/reoxygenation-induced apoptosis. Ventricular myocytes from adult or neonatal rates were cultured and subjected to hypoxic conditions (0.2% O₂) for 18 or 24 hrs (adult/neonatal) followed by a reoxygenation period (21% O₂) of 24 hrs. Hypoxic condition was proved by HIF1-alpha expression using western blots. Apoptotic cell analysis was determined by using the ViaCount assay (Guava Technologies). In adult myocytes, hypoxia caused 34% and hypoxia/reoxygenation 31% apoptotic cells. Application of DM-CHX (5µM) resulted in 22% (hypoxia) and 11% (hypoxia/reoxygenation) apoptotic cells. In neonatal myocytes, under both conditions 64% apoptotic cells were analysed, reduced to 34% after DM-CHX (5 µM) treatment. As positive control, caspase-inhibition and cyclosporin A showed apoptosis inhibition in both types of myocytes. Hypoxia (2.5-fold) and hypoxia/reoxygenation (1.4-fold) caused injury of adult myocytes measured by relative lactate dehydrogenase activity, which was reduced by DM-CHX (5 µM) treatment. Our results suggest that DM-CHX, a specific inhibitor of FKBP38, reduces apoptosis in cardiomyocytes in a dose-dependent manner. Such a specific drug could be used to decrease the loss of myocytes after damaged injury resulting in an improved cardiac function.

2E_06_S

NF-kB functions the diversity of cellular IGF-I/IGFBP-1 expression by hypoxia in Tibetan Plateau mammals

X. Q. Chen, S. J. Wang, Y. Liu and J. Z. Du

dujz@cls.zju.edu.cn

Ochotona curzoniae, Microtus oeconomus and Myospalax baileyi are all native mammals that reside at Qinghai-Tibetan plateau in China and well acclimatized to environmental hypoxia. The present paper addresses the NF-kB’s, a nuclear transcriptional factor, involvement in hypoxia stress-induced diversity of IGF-I/IGFBP-1 expression in hepatic and brain cells of Tibetan Plateau mammals. The IGF-I/IGFBP-1 from the prefrontal cortex and the liver cells was tested 6 h after hypoxia exposure (by CoCl2 injection i.p. 20, 40 mg/kg or by normobaric hypoxia, 16.0%, 10.8%, 8.0 %O2) of the Plateau native mammals and mice. PDTC, an inhibitor of NF-?B, was used and preinjected before the hypoxia to evaluated NF-kB action. The results showed that 1) the IGF-I expression in mice hepatic cells of M. oeconomus and M. baileyi markedly increased after the hypoxia exposure, but there was no response in the liver of O.curzoniae; 2) the IGFBP-1 expression in mice hepatic cells of O. curzoniae and M. baileyi markedly enhanced, but no response occurred in M. oeconomus after the hypoxia; 3) PDTC pretreated before hypoxia reversed the hypoxia-enhanced IGF-I in M. oeconomus and M. baileyi; 4) PDTC treatment also reversed the hypoxia-enhanced IGFBP-1 in O. curzoniae and M. baileyi; 5) hypoxia increased the IGF-I mRNA in brain of M. oeconomus and O. curzoniae but not of mice; 6) hypoxia did not induce changes of IGF-I levels in the brain cells of both plateau mammals and laboratory mice. The data suggest that 1) different pattern in IGF-I/IGFBP-1 expression induced by hypoxia represents a diversities in hormone regulation and cell protection from damage in Tibetan native mammals; 2) NF-?B mediates the transcription of IGF-I/IGFBP-1 in liver cells subjecting to hypoxia; Together, the diversity of target-gene phenotype expression may contribute to the multi-model in cell protection from hypoxia damage.Acknowledgement: This work was supported by the NSFC: Projects (No. 30393130;30470648; 30570227), and by The National Basic Research Program “973” No. 2006CB504100).

2F_01_S

Thylakoid Proteases in Higher Plants - Roles in Protein Quality Control

Zach Adam

The Hebrew University, The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Rehovot 76100, Israel. E-mail: zach@agri.huji.ac.il

The dependence of photosynthesis on light is obvious. The higher the light intensity, the higher is the rate of photosynthesis, up to a level where light energy is no longer limiting and photosynthesis remains constant. However, further increase in the intensity of light may lead to a decrease in photosynthesis rate, a phenomenon known as ‘photoinhibition’. Photoinhibition is attributed to oxidative damage, primarily to photosystem II (PSII) and its reaction center protein D1. A number of mechanisms have been evolved during evolution to minimize oxidative damage, but if PSII is damaged after all, a PSII repair cycle operates to allow photosynthesis to proceed. A key component of this cycle is the proteolytic removal of damaged D1 protein, prior to its replacement by a newly synthesized one. Degradation of the D1 protein has been a central question in the field of photosynthesis for the past 20 years or so, but only in recent years the identity of the proteases involved has started to unravel. Recombinant FtsH, a thylakoid ATP-dependent metalloprotease, was first shown to participate in D1 degradation in an in vitro study. Later on, in vivo analysis of Arabidopsis FtsH mutants revealed that they were more sensitive to photoinhibition than wild type, and that damaged D1 protein was stabilized in them. Further analysis of different mutants suggested that the chloroplast FtsH complex is composed of two essential types of subunits, each one of them is encoded by two redundant genes. More recently, analysis of knock-down mutants of the lumenal serine protease Deg1 suggested that this protease is also involved in the process of D1 degradation. Wider implications to questions of chloroplast biogenesis and maintenance will be discussed.

2F_02_S

Protein folding, quality control and degradation in the ER: the role of N-glycans

Tatiana Solda, Tito Cali, Carmela Galli, Maurizio Molinari

Institute for Research in Biomedicine, Bellinzona, Switzerland, maurizio.molinari@irb.unisi.ch

The ER is the site of folding and assembly of proteins destined for the plasma membrane, the secretory and endocytic organelles and the extracellular space. Most of the proteins synthesized in the ER are covalently modified by co-translational addition of pre-assembled glucose₃-mannose₉-N-acetylglucosamine₂- (Glc₃-Man₉-NAcGlc₂) core oligosaccharides. Protein-bound oligosaccharides are exposed to several ER-glycanases that sequentially remove terminal glucose or mannose residues. Rapid generation of a mono-glucosylated (Glc₁-Man₉-GlcNAc₂) trimming intermediate is required to enter the calnexin chaperone system in which protein folding progresses with highest efficiency. Removal and re-addition of the innermost glucose residue activate cycles of dissociation/re-association with calnexin that may facilitate, and in some cases is required for acquisition of the polypeptide’s native structure. Slower removal of terminal a1,2-bonded mannose residues from N-linked glycans occurs upon persistent polypeptide retention in the ER, which is symptom of defective folding. Substrate de-mannosylation eventually interrupts futile folding attempts, results in substrate exclusion from the calnexin chaperone system and promotes retro-translocation into the cytosol for degradation operated by the 26S proteasome. De-glucosylation and de-mannosylation activities must be tightly regulated because the N-glycan composition will determine if the associated protein will be subjected to folding-attempts in the ER lumen or if it will be retro-translocated into the cytosol and degraded.

2F_03_S

Protein Quality control and Degradation at the Endoplasmic Reticulum

Robert Gauss, Ernst Jarosch, Christian Hirsch, and Thomas Sommer

Max-Delbrück-Center for Molecular Medicine, 13092 Berlin, Germany

A quality control system surveys the lumen of the endoplasmic reticulum (ER) for terminally misfolded proteins. Polypeptides singled-out by this system are ultimately degraded by the cytosolic ubiquitin proteasome pathway. This process is termed ER-associated protein degradation (ERAD). A central ERAD component is the ubiquitin ligase Hrd1/Der3. This ligase forms a complex with its partner protein Hrd3 and with the ER-membrane protein Der1. Our data imply that Hrd3 is the major substrate receptor of this heterogenic ligase complex in the ER-lumen. Although Hrd3 and Der1 bind to soluble substrate proteins independently, both proteins are essential to trigger substrate dislocation. At the cytosolic face of the ER the Hrd1-complex associates with the AAA-ATPase Cdc48/p97. Cdc48p binding depends on it’s membrane receptor Ubx2, but most importantly also on substrate processing by the Hrd1-complex, suggesting that ubiquitination precedes substrate mobilization by the Cdc48/p97-complex.In addition, we were able to detect an interaction between the ER quality control lectin Yos9p and Hrd3p. We have identified designated regions in the luminal domain of Hrd3p that interact with Yos9p and Hrd1p. Binding of misfolded proteins occurs via Hrd3p, suggesting that Hrd3p recognises proteins which deviate from their native conformation while Yos9p ensures that only terminally misfolded polypeptides are degraded.

2F_04_S

Protease and chaperone functions in the maintenance of mitochondrial protein homeostasis

Claudia Leidhold, Tom Bender, and Wolfgang Voos

Institute for Biochemistry and Molecular Biology, University of Freiburg, Herrmann-Herder-Str. 7, D-79104 Freiburg, Germany

Apart from supplying ATP, mitochondria are involved in crucial biosynthetic and signaling processes in a eukaryotic cell. Mitochondrial protein homeostasis is determined by the import of newly synthesized proteins and proteolytic removal of excess or damaged polypeptides. Damaged polypeptides, generated under environmental stress conditions, are first recognized by chaperones, stabilized and refolded to the functional state. If this fails, the proteins are transferred to the proteolytic system for their removal. The coordinated activities of chaperones and proteases form a protein quality control system that is required for the maintenance of organellar function. Mitochondrial proteases belong to the AAA+ protein family and can be separated into soluble and membrane-integrated types. We used a proteome analysis of isolated mitochondria to determine the native substrate selectivity of these proteases. We were able to identify a group of specific substrates for the matrix protease Pim1 that were distinguished by an intrinsic low structural stability and the presence of small molecule cofactors. Cells lacking mitochondrial proteases showed a higher sensitivity to high levels of reactive oxygen species (ROS). A specific subgroup of mitochondrial proteins showed enhanced degradation rates in the presence of ROS. Enzymes containing Fe/S cluster exhibited a high sensitivity to increased ROS levels. Interestingly, proteins that belonged to the ROS detoxification system showed the highest relative degradation rates. We conclude that the protein quality control system contributes prominently to the maintenance of mitochondrial protein functions under stress conditions.

2F_05_S

Genetic dissection of the protein quality control in Escherichia coli

Elena García-Fruitós¹, Monica Martínez-Alonso¹, Nuria Gonzalez-Montalbán¹, Minoska Valli^2,3, Diethard Mattanovich^2,3, Antonio Villaverde^1*

3 School of Bioengineering, University of Applied Sciences FH-Campus Vienna, Austria.

Keywords: E. coli; proteolysis; protein folding; inclusion bodies; chaperones

Abstract:

The protein quality control system is an evolutionary conserved complex mechanism based on a network of cellular proteins with overlapping foldase, disaggregase and protease activities. In bacterial cells, the coordinated activity of all these elements promote proper protein folding or digestion of folding-reluctant, potentially toxic species, what is expected to keep misfolding-prone protein species in a soluble state and cells free from aggregates. However, recent insights on the biology of protein misfolding and aggregation strongly suggest that solubility and conformational quality are not matching events, since at least in some examples, protein aggregates might contain properly folded species. By using Escherichia coli cell models, we have finely explored both protein solubility and quality, through the fluorescence emission of green fluorescent protein (GFP) engineered variants, in mutant cell strains lacking defined components of the quality control system, including main chaperones, small heat-shock proteins and proteases. In absence of either chaperones DnaK, ClpB or ClpA and proteases ClpP or Lon, GFP-producing cells are significantly more fluorescent than wild type cells (up to more than two fold), while the solubility of GFP is clearly higher in wild type cells exhibiting a fully functional quality control network (up to around two fold). In all the identified mutants, the enhanced emission of recombinant cells is clearly linked to an elevated intracellular content of highly fluorescent GFP molecules, resulting from inhibition of its proteolytic degradation and the significant expansion of its half life (from 2 to up to 7 h, at 37oC). Interestingly, the excess of functional protein is overstocked as highly fluorescent inclusion bodies containing properly folded protein species. Overall, these results indicate that the E. coli quality control system is governed by an over-committed, chaperone-mediated proteolytic machinery that acts on protein species that are either functional or can reach functional forms when proteolytically stabilized. Intriguingly, the occurrence of molecular determinants of aggregation does not require complete protein unfolding, and in fact, solubility and fluorescence emission are inversely correlated. Therefore, selected genetic deficiencies in the quality control system dramatically enhance the intracellular pool of biologically active although insoluble, misfolding-prone proteins, a fact that might be specially relevant in the context of high quality recombinant protein production.

2F_06_S

Lectin-deficient calreticulin retains full functionality as a chaperone and quality control component during the biogenesis of class I histocompatibility molecules.

Breanna S. Ireland, Ulf Brockmeier and David B. Williams

Depts. of Biochemistry and Immunology, University of Toronto, Toronto, Canada, M5S 1A8, david.williams@utoronto.ca

Calreticulin (Crt) is a soluble chaperone of the endoplasmic reticulum (ER) that interacts with newly synthesized glycoproteins through a lectin site with specificity for Glc₁Man₉GlcNAc₂ oligosaccharides as well as through a polypeptide binding site that recognizes non-native protein conformers. The relative contribution of each site to the overall functions of Crt remains unknown. To address this issue, we created two point mutants, D317A and Y128A, that ablate the lectin function of Crt but do not alter its tertiary structure. We then examined their abilities to support the biogenesis of mouse class I histocompatibility molecules. Class I molecules function to present peptide antigens to cytotoxic T cells. They consist of a glycosylated transmembrane heavy chain, a soluble subunit termed b ₂-microglobulin and an 8-9 residue peptide ligand. In Crt-deficient cells, the surface expression of class I molecules is reduced 2- to 3-fold, loading of peptide ligands is inefficient, and peptide-deficient class I molecules are prematurely exported from the ER (defective quality control). We expressed wild type Crt as well as both lectin-deficient Crt mutants in Crt-deficient cells. Remarkably, the lectin-deficient mutants were just as effective as wild type Crt in upregulating class I surface expression, enhancing peptide loading and preventing premature export from the ER. The mutants were also capable of binding to many newly synthesized glycoproteins in addition to class I. We conclude that in the absence of lectin-based interactions, Crt can utilize polypeptide-based interactions to effect its chaperone and quality control functions.

2G_05_S

Function of DnaK in Streptococcus intermedius

Toshifumi Tomoyasu, Atsushi Tabata and Hideaki Nagamune

e-mail: tomoyasu@bio.tokushima-u.ac.jp

Streptococcus intermedius is an anaerobe and belongs to the anginosus group of streptococci (AGS), which constitute a part of the normal flora of the human oral cavity as well as the upper respiratory, gastrointestional, and female urogenital tracts. AGS are recognized as opportunistic pathogens that cause purulent infections and abscess formation. We have previously reported that DnaK chaperone controls the expression of flagella and several pathogenic factors in Salmonella Typhimurium. Therefore, we constructed a dnaK null mutant from S. intermedius in order to investigate the possible role of DnaK in pathogenicity. The generation time of a dnaK null mutant from S. intermedius was approximately twice that of the parent strain. Similar to other gram-negative bacteria, the dnaK null mutant exhibited a thermosensitive phenotype and could not grow above 40°C. However, the dnaK null mutant did not show acid and H₂O₂ sensitivity, which is characteristic of gram-negative bacteria. Interestingly, GroEL accumulation was observed in the dnaK null mutant. The genome sequences from AGS revealed that the heat shock response, including expression of the groESL operon, appears to be controlled by the HrcA heat shock gene transcriptional repressor. Our result suggests that DnaK might regulate the activity or cellular amount of HrcA. Neither the dnaK mutant nor the parent strain showed a significant difference with regard to the activity of cytolysin (Intermedilysin) and hyaluronidase, and in the ability to form biofilms. These data indicate that DnaK in S. intermedius plays a role in the fundamental functions for living (e.g., growth, thermoresistance, and heat shock regulation) but has less functionality in the modulation of expression of pathogenic factors.

2G_06_S

Module 2 – Poster lectures:

2A_01_P

Osteoprotegerin as an Anti-Apoptotic Protein

Nina C. Dempsey, Claire Hunter-Lavin, Michael J. Marshall & John H.H. Williams

n.dempsey@chester.ac.uk

The role of OPG in bone turnover through its interaction with RANKL is well established, however this protein has now been shown to have anti-apoptotic properties which may contribute to the survival of cancer cells. As confirmation of this potential role we show data that OPG can interact with both TRAIL and TNF? using ELISA and BIAcore. We have then demonstrated that OPG will protect cells against TRAIL or TNF? induced apoptosis.

We have then focused on the release of OPG under stress conditions from MG63 cells and looked at the existence of OPG feedback mechanisms. We show that a number of stressors that induce apoptosis also result in an increase in the release of OPG from cells. These stressors include – UV exposure and exposure to TNF? or TRAIL. The increase in release of OPG occurs prior to indicators of apoptosis – caspase 3 activation or Annexin V binding. Interestingly, induction of apoptosis by elevated temperature coincides with a total cessation in OPG release.

Production of OPG can be regulated by a feedback mechanism as release can be increased by lowering the ambient concentration of OPG, and conversely that by increasing this ambient concentration, the release of OPG is inhibited. The changes are mirrored by alterations in gene expression.

We discuss the potential role of OPG in the treatment of tumours and strategies that can be used to bypass this protection.

2A_02_P

Hyperosmotic stress response

Roberta R Alfieri, Mara Bonelli, Andrea Cavazzoni, Angelo F Borghetti, Pier Giorgio Petronini

Dipartimento di Medicina Sperimentale Universita degli Studi di Parma Via Volturno 39 Parma E-mail: roberta.alfieri@unipr.it

Many types of mammalian cells can survive a moderately hypertonic environment due to a specific adaptation process that results in the cellular accumulation of compatible osmolytes. This adaptation process, involves early responses, occurring over milliseconds to minutes, and later responses, requiring hours to days. The virtually instantaneous reduction in cell volume due to the osmotic efflux of water induced by acute hypertonic stress is rapidly corrected by what is referred to as regulatory volume increase (RVI). This early process is mediated by pre-existing ion transport systems that produce increases in the intracellular concentrations of potassium, sodium and chloride ions, and the accompanying influx of water causes RVI. The later phase is characterised by increased production of heat shock proteins (HSPs) and either the synthesis or the uptake and cellular accumulation of compatible osmolytes. In mammalian cells the latter include neutral amino acids or their derivatives, polyols such as sorbitol and myo-inositol, and methylamines such as betaine. The usual explanation of this phenomenon is the need to replace the early cellular accumulation of inorganic ions with small organic molecules that do not affect cell function even at relatively high intracellular concentrations. Accumulation of compatible osmolytes within the cell thus maintains intracellular water homeostasis without impairing normal biochemical functions such as protein synthesis. Cells do not adapt and die by apoptosis when hypertonic medium has been depleted of these molecules. Compatible osmolytes thus enable cells to survive under hypertonic conditions, protecting them from apoptosis and modulating the adaptive response.

2A_04_P

Glutathione S-Transferase pi regulates UV-induced JNK signaling in SH-SY5Y cells

M Castro-Caldas^1,2, E Rodrigues¹, MC Lechner¹, MJ Gama¹

2SABT. Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa

Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a key event in neuronal apoptosis. Previous studies demonstrated that in SH-SY5Y cells UV-induced apoptosis is associated with activation of JNK. The cellular mechanisms underlying the control of JNK activity before and immediately after stress are not completely understood. Under resting conditions the basal activity of JNK is low, since JNK is kept inactive by the presence of one or more repressors. Inactivation of JNK may be mediated by binding through protein-protein interactions to non-substrate proteins, including Glutathione S-Transferase pi (GSTpi). GSTpi belongs to a multigene family of isozymes catalyzing detoxification reactions. Although it has previously been shown that over expression of GSTpi protects cells from JNK-mediated apoptosis, the mechanisms underlying regulation of JNK signaling by GSTpi in neuronal cells have never been described. In this work, SH-SY5Y cells were treated with UV to evaluate the regulation of JNK signaling by GSTpi. The relative concentrations of the GSTpi, p-JNK/JNK and apoptotic proteins were estimated by Western blot. Direct interaction of GSTpi and JNK was determined by co-immunoprecipitation assays. Evaluation of GSTpi dimers and multimeric complexes formation was performed by SDS-PAGE under non-reducing conditions. Our results show that UV treatment induces apoptosis in SH-SY5Y cells and the transient activation of JNK. Furthermore, the increase of JNK enzymatic activity correlates with changes of GSTpi-JNK complexes and the concentration of GSTpi multimer forms. Taken together our results suggest that GSTpi may act as a regulator of the UV-induced cellular stress response, controlling JNK activity by protein-protein interactions.

2A_05_P

THE Role of hsp72 in neuronal cell survival and the resistance of neurone-like SH-SY5Y cells to apoptotic insults

Cheng, L., ^1* Smith, DJ., ¹ Nagley, P¹ and Anderson, RL²

*Lesley.Cheng@med.monash.edu.au

Heat shock proteins have been implicated in neuronal cell survival. We studied here the anti-apoptotic effects of heat-inducible hsp72 in the human SH-SY5Y neuroblastoma cell line, propagated in an undifferentiated form and able to be differentiated into neurone-like cells using retinoic acid with brain-derived neurotrophic factor. Mild heat stress (43C for 30 min) was applied to induce hsp72, subsequently referred to as thermal pre-conditioning treatment. It was observed that thermal pre-conditioning protects cells against apoptosis induced by a subsequent treatment with staurosporine (50 nM). Neurone-like SH-SY5Y cells displayed reduced Bax activation, cytochrome c release and nuclear fragmentation when thermally pre-conditioned compared to non-pre-conditioned control cells (all monitored by immunocytochemistry and confocal microscopy). The suggestion that hsp72 may be involved in blocking apoptosis and that the block by hsp72 may be upstream of Bax was tested by constructing stable transfectants over-expressing hsp72 (5YH72.1). Such cells maintained levels of hsp72 comparable to those seen in untransfected undifferentiated SH-SY5Y cells exposed to thermal pre-conditioning; similar levels of hsp72 were also found in neurone-like untransfected cells without heat shock, conditions that also induce hsp72. 5YH72.1 cells showed enhanced thermotolerance, at significantly higher temperatures than neurone-like untransfected cells (themselves more thermotolerant than their undifferentiated counterparts). Moreover, neurone-like 5YH72.1 cells treated with 50 nM of staurosporine failed almost completely to display Bax activation and nuclear fragmentation. Undifferentiated 5YH72.1 cells were also protected but to a lesser extent against the molecular effects of staurosporine treatment. The data support the proposition that hsp72 is responsible for the thermoprotection observed in SH-SY5Y cells. Further, hsp72 acts upstream of the mitochondria to prevent apoptosis in these cells when expressed in moderately high quantities.

2A_06_P

Upregulation of anti-apoptotic Bcl-2 by a novel endoplasmic reticulum protein sigma-1 receptor

Teruo Hayashi and Tsung-Ping Su

Intramural Research Program, NIDA, NIH, DHHS, 333 Cassell Drive, Baltimore, MD 21224. thayashi@intra.nida.nih.gov

Extreme cellular stress induces apoptosis, a deliberate life-relinquishment of a cell. Bcl-2 serves as a powerful antidote against apoptotic cell death by preventing the mitochondrial permeability transition that causes the release of caspase activators. Although the level of Bcl-2 has been shown to be altered under a variety of cellular stresses, signaling pathways that regulate the Bcl-2 expression are not fully understood. Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) proteins that are activated by steroids and psychotropic drugs. Sig-1Rs express highly in the nervous system as well as in carcinomas. Activation of Sig-1Rs is known to promote a robust neuroprotective action, whereas inhibition of Sig-1Rs promotes cell death such as seen in cancer cells. Recent studies have proposed, therefore, the Sig-1R as a potential therapeutic target in treatments of ischemia and cancer. Here, we reported that the Sig-1R is a novel ER protein regulating Bcl-2 expression. Sig-1Rs co-localized with Bcl-2 at the ER, but not at mitochondria in CHO cells. Overexpression of Sig-1Rs significantly increased Bcl-2 proteins in mitochondria, whereas knockdown of Sig-1Rs by siRNAs caused a prominent downregulation of Bcl-2. RT-PCR and Northern blotting revealed that knockdown of Sig-1Rs downregulated the bcl-2 mRNA, indicating a transcriptional activation and/or mRNA degradation of bcl-2 by Sig-1Rs. In keeping with these findings, knockdown of Sig-1Rs potentiated cell death when CHO cells were under apoptotic stimuli. Our findings suggest that the novel ER protein Sig-1R is intrinsically regulating the level of mitochondrial Bcl-2, and, thus, Sig-1R agonists such as pregnenolone sulfate and DHEAS may exhibit cell protective action, a least in part, by increasing the expression and thus augmenting the anti-apoptotic activity of Bcl-2. (Sponsored by IRP/NIDA/NIH/DHHS)

2A_07_P

HtrA2 is up-regulated in the heat-stressed rat testis

TETSUO HAYASHI, NOBUYUKI ISHII, TOSHIYA TERAO, MAKOTO MOROZUMI AND TAKUMI YAMADA

Department of Urology, Saitama Medical Center, Saitama Medical University, Kawagoe, Saitama, Japan

Aim: The aim of the present study was to elucidate the role of the high temperature requirement A 2 (HtrA2) for the germ cell loss in the heat-stressed testis.

Methods: We examined the expression of HtrA2, caspase-9 activity and proteolytic activity of HtrA2 in the rat testis and their in vivo responses to experimental cryptorchid treatment.

Results: Northern analysis revealed the expression of HtrA2 mRNA peaked at days 1 and 7 after cryptorchid treatment. While the expression of HtrA2 mRNA was recognized in the spermatogonium, spermatocytes and some spermatids in normal adult rat testis, the experimental cryptorchidism treatment resulted in a marked increase in its signal intensity in spermatocytes and some spermatids and the layers of spermatogonium and early primary spermatocytes became negative at days 1 and 7 after the treatment. However, the spermatogonium, Sertoli cells and interstitial cells appeared to have strong intensities at days 14, 28 and 56 after the treatment. Western analysis revealed the expression of HtrA2 protein peaked at days 2 and 28, Caspase-9 activity peaked at day 2 and HtrA2 proteolytic activity peaked at day 28. Consequently, the first peak of HtrA2 mRNA expression was followed by the peak of caspase-9 activity and the second peak was followed by the peak of proteolytic activity.

Conclusion: These findings suggest the probabilities that the heat stress results in germ cell death by caspase-independent manner with the elevation of proteolytic activity of HtrA2 as well as caspase-dependent manner with the elevation of caspase-9 activity.

2A_08_P

Functional Studies on ubiquitin receptor protein, hFAF1, in stress responses.

Jejean Lee, Young-Mee Kim, Kong-Joo Lee

kjl@ewha.ac.kr 

Human Fas-associated protein, hFAF1, was identified as Fas-associating molecule and a member of the apoptosis signaling complex. We identified that hFAF1 acts as a scaffolding protein by unveiling the interacting proteins and newly found N-terminal ubiquitin-associated (UBA) domain. N-terminal UBA domain was identified to recruit K48- and K63-linked polyubiquitinated proteins and plays a role to accumulate the ubiquitinating proteins as a ubiquitin receptor. One ubiquitin like (UBL) domain interacts with Hsp70 and negatively regulates its chaperone activity. C-terminal ubiquitin regulatory X (UBX) domain was identified to interact with AAA ATPase p97/VCP which is involved in the ubiquitin-proteasome pathway. These interactions of hFAF1 to two chaperone proteins (Hsp70 and VCP) suggest that FAF1 as a ubiquitin receptor plays important roles in stress response and apoptotic cell death. In this study, we will demonstrate the regulation mechanism by examining the post-translational modifications using proteomic tools, and the biological functions by examining the effects of VCP binding defect mutants and RNAi of hFAF1 in response to stress. This can suggest the biological role and regulation of ubiquitin receptor hFAF1 in stress-induced ubiquitin-proteasome system. [Supported by KOSEF NCRC for CCS & DDR and FPR05A2-480. J Lee and YM Kim supported by BK21]

2A_09_P

PPARg agonists and HSP70 renders the resistance to apoptosis in g-irradiated cancer cells

Piotr Pierzchalski¹, Agata Krawiec¹, Wiesław Pawlik¹, Maciej Gonciarz²

2 Endoscopy Unit, St. Barbara District Hospital, Plac Medyków 1, 41-200 Sosnowiec, POLAND

The involvement of peroxisome proliferator-activated receptors (PPARs) in the cancer cell apoptosis is a generally accepted fact. However, some reports indicate that the activation of PPARg is directly responsible for carcinogenesis.

It is well known that the high level of heat shock proteins (HSPs) in cancer cells is associated with metastasis, the poor prognosis and the resistance to radio as well as chemotherapy. HSP70 as a part of the most important systems for maintaining the viability of the cell, is known to counteract against the apoptosis. We report here the involvement of HSP70 in anti-apoptotic action of activated PPARg in g-irradiated human colon cancer cells.

We have used Caco-2 cells (human colon adenocarcinoma) as an experimental model. In this system PPAR-g agonists induced nuclear translocation of PPAR-g as well as HSF-1. This translocation was followed by the increase of HSP70 mRNA and protein expression.

Cells subjected to g-radiation (photons) with therapeutic dose of 2,5 Gy, manifested pattern of PARP degradation typical for apoptosis, showing both the native 112 KD and digested 85 KD forms. It suggests activation of caspases 3 or 6. Stimulation of the cultures with PPARg agonists prior to the irradiation eliminated altogether the process of PPAR-g nuclear translocation and PARP degradation. PPARg remained in the complexes with AKT-1 in cytoplasmic as well as in nuclear pool. However this treatment did not affect HSF-1 translocation and HSP70 expression.

According to our elucidation, in g-irradiated cells nuclear translocation of PPARg is abolished and PPARg-AKT-1 complexes are conserved in which PPARg remains insensitive for its agonists treatment. Most likely, at the same time PPARg agonists directly activate HSP 70. The process is undisturbed by the g-irradiation what renders the colon cancer cells resistance to apoptosis.

2A_10_P

Relationship between apoptosis and HSP70 expression in lymphocytes exposed to stress mediators

Sapozhnikov A.M., Kovalenko E.I., Murashko D.A., Alekperov E.A., Shustova O.A.

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia, amsap@mail.ru

It has been shown that adrenaline in contrast to glucocorticoids can activate immune system. However existing data indicate that catecholamines like glucocorticoids induce apoptosis in lymphocytes. Earlier we demonstrated that lymphoid cell apoptosis is accompanied by increase of HSP expression. In this work we carried out a study of effect of adrenaline and dexamethasone on apoptosis and HSP70 expression in cultures of mouse lymphocytes. Obtained results confirmed apoptogenic effect of catecholamine and dexamethasone. However, the stress mediators had opposite effects on level of HSP70 expression: adrenaline enhanced intracellular content of the protein whereas dexamethasone had small but significant inhibitory action on HSP70 expression. The catecholamine-induced increase of HSP70 expression was mediated by a -adrenoreceptor because the effect was suppressed by phospholipase C inhibitor but was not decreased by PKA inhibitor. In addition, we have revealed that a -adrenergic antagonist prazosin reduce adrenaline-induced HSP70 expression while b -adrenergic antagonist propranolol decrease the apoptogenic effect of catecholamine. Phenylephrine – specific a -adrenergic agonist had no apoptogenic effect in contrast to adrenaline interacting with both a - and b -adrenoreceptors. We suppose that the difference between immunomodulating activity of catecholamines and glucocorticoids may be connected with their opposite influence on lymphocyte HSP system. The results suggest that effect of catecholamines on apoptosis and HSP70 expression are initiated by the same hormonal signal perceived by means of different adrenoreceptors.

This study was supported by grants from RFBR (06-04-49568) and BTEP/ISTC (73/2627).

2A_11_P

Expression of heat shock proteins in primary porcine myotubes exposed to stressors

Straadt, I. K.^1*; Young, J. F.¹; Bertram, H. C.¹; Gregersen, N.²; Bross, P.²; Oksbjerg, N.¹

*Cor. author: ida.straadt@agrsci.dk

Stress exposure to animals prior to slaughter is causing big variations in the meat quality. Using primary porcine myotubes as a model, exposure to different stressors was investigated. When myotubes were exposed for 1 h to 50-250µM H₂O₂ the peak in expression of mRNA for HSP70 and HO1, measured 18 h after stress exposure, was seen at a concentration of 200 µM H₂O₂. At 250 µM the expression decreased substantially, indicating that the cells were no longer capable of expressing heat shock proteins at a high level. Also the appearance of the cells evidenced the toxic effect of H₂O₂ levels above 200 mM. When testing the viability this was confirmed as the survival of myotubes exposed to more than 100 µM H₂O₂ for 1 h showed a decreased cell viability determined by WST-1. Based on these observations a level of 100 µM H₂O₂ was chosen for investigating the development in expression levels of heat shock proteins over a time period in the myotubes. A significant increase in expression of both HSP70 and HO1 mRNA was observed after exposure to H₂O₂ for 1 h and measuring expression up to 18 h after exposure. No change in the cell viability determined by WST-1 was observed when the myotubes were exposed to heat (42° C or 45° C) for 1 h. However, comparing cells exposed to 42° C and 45° C the expression was approximately 10 times higher when exposed to 45° C. No change in the expression level of HO1 mRNA was observed when exposed to heat. Hence, primary porcine cells are more sensitive to H₂O₂ compared to myotubes and muscle cell lines from other species. In contrast to this, primary porcine muscle cells are not very sensitive to heat stress when determining the cell viability by WST-1.

2A_12_P

Apoptosis and Heat Shock Protein HSP70 in aged mammalian oocytes.

P. Esponda ¹ and H. Díaz ²

2 Facultad de Ciencias. Universidad de Valparaíso. Valparaíso. Chile

The fertile life of the mammalian is short and a few hours after ovulation it loses its capacity to be fertilized. Ageing affect oocyte fertilizing ability and aged females produce numerous infertile and/or abnormal oocytes. We used normal and aged mouse oocytes to analyze initial apoptosis (using Anexin V), late apoptosis (using the TUNEL method) and the presence of the stress protein HSP70 (using antibodies and RT-PCR). We induced the ovulation by hormones (PMSG-HCG) to collect oocytes from young females (3 months old). Gametes were also aged in the oviduct after ovulation during 20 hours (postovulatory ageing) and other oocytes were collected from 12-15 months old females (preovulatory ageing).

Anexin V showed that a high percentage (39.2%) of initial apoptosis appeared in oocytes ovulated by old females, and in oocytes aged in the oviduct during 20 hours (31.6%). TUNEL results showed that the high percentages (56.5%) of apoptosis occurred when oocytes were aged in the oviduct during 20 hours. Oocytes from young or old females showed a similar degree of damage (12.9-16.1%). Nevertheless, young oocytes recovered from females that were not treated with hormones, show a lower percentage of apoptosis (3.7%). TUNEL procedure was also applied to histological sections from ovaries of different ages (3; 11; 14 and 24 months old). Results showed that apoptotic cells were progressively increased in the insterticial tissue of the organ.

Normal oocytes do not showed the presence of the stress protein HSP70, but this protein appeared in the cytoplasm after an exposure to 40oC during 4 hours, and in oocytes after postovulatory ageing. RT-PCR showed HSP70 gene expression in aged oocytes.

These results indicated that ageing increase apoptosis of the mouse oocyte and of the ovarian tissue. Apoptosis and HSP70 are principally produced by environmental conditions, as the long storage of the oocyte in the oviduct or the employ of hormones used to induce ovulation.

2A_13_P

Stress hormones induce in cancer cells ATP depletion via perturbation of oxidative phosphorylation

Eliezer Flescher

Tel Aviv University, Tel Aviv 69978 Israel, flascher@post.tau.ac.il

The jasmonate family of plant stress hormones exhibits selective cytotoxic activities towards cancer cell lines as well as leukemic cells from chronic lymphocytic leukemia patients. The aim of this project was to elucidate the jasmonate mechanism of action. We found that jasmonates induce a rapid depletion of ATP in cancer cells, preceding any signs of cell death. Furthermore, we found a positive correlation between the susceptibility of a given cell type to the cytotoxic effect of jasmonates and the degree of ATP depletion induced in that cell. The two major sources of cellular ATP, oxidative phosphorylation and glycolysis, determine the steady state levels of ATP. Experiments using modulators of ATP synthesis via glycolysis or oxidative phosphorylation suggest that the latter is the pathway suppressed by jasmonates. Consequently, the direct effects of jasmonates on mitochondria were evaluated. Jasmonates induced cytochrome c release and swelling in mitochondria isolated from cancer cells but not from normal ones. Thus, the selectivity of jasmonates against cancer cells is rooted at the mitochondrial level, and probably exploits differences between mitochondria from normal versus cancer cells. The permeability transition pore complex (PTPC) regulates movement of compounds across the mitochondrial membrane. Abnormally long opening of this pore can be associated with cytochrome c escape into the cytosol, resulting eventually in cell death. Jasmonate-induced release of cytochrome c from mitochondria isolated from cancer cells was inhibited by inhibitors of PTPC opening, suggesting that the mitochondrial permeability transition induced by jasmonates is PTPC-mediated. These findings position jasmonates as promising anti-cancer drugs acting via energetic depletion in neoplastic cells.

2A_14_P

Oxidative stress and apoptosis in carcinogenesis: molecular players and interactions

Claudia Campanella1, Nella M. Ardizzone1, Antonella Montalbano1, Antonella Marino Gammazza1, Anna Ribbene1, Anna M. Czarnecka2, Valentina Di Felice1, Marianna Bellafiore1, Giovanni Zummo1, Francesco Cappello1, Everly Conway de Macario3, Alberto J. L. Macario3

1Department of Experimental Medicine, University of Palermo, Italy; 2Department of Genetics, University of Warsaw, Poland; 3Center of Marine Biotechnology, University of Maryland, Baltimore, MD, USA.

Oxidative stress (OS) and apoptosis are major determinants of cell fate in normal and tumor cells. The connection between OS and apoptosis is not fully understood; the molecular networks involved have not been elucidated yet. Our work's objectives are to identify network components and determine their interactions and the consequences of the interactions in tumor cells. The long-term goal is to understand at the molecular level the contribution of OS and apoptosis to carcinogenesis and thus identify precise targets for anti-tumor therapy. We used a tumour cell line (NCI-H292 human airway mucoepidermoid carcinoma) and measured, before and after OS induction with various doses of H2O2, molecules known or suspected to participate in the cell's response to OS, including apoptosis: Hsp60, Hsp10, Hsp70, p53, procaspase C3 (p-C3), caspase 3 (C3), and p21. Various complementary methods were applied to assess cell viability and apoptosis, and to determine the cellular localization of the molecules investigated and their quantities at the levels of mRNA and protein. Occurrence of the bimolecular complexes Hsp60/p-C3 and Hsp60/p53 was also investigated before and after OS induction. Several findings were made; most conspicuous of all were the presence of Hsp60/p-C3 complexes and the lack of C3 after OS induction despite cell progression through apoptosis. The link between Hsp60/p-C3 complex formation and C3 absence is currently under study.

2A_15_P

Interactions between important regulatory proteins and the stress response protein, ?B crystallin

Joy G. Ghosh†, Ananth K. Shenoy Jr.†, & John I. Clark†‡.

Dept. of Biological Structure† & Dept. of Ophthalmology‡, Univ. of Washington, Seattle, WA 98195, USA.

?B crystallin, the archetype of small heat shock proteins (sHSPs), is an 'unfolding response protein' that recognizes, binds, and stabilizes structurally compromised proteins during or after stress. Protein pin arrays identified interactive sequences in ?B crystallin for twelve regulatory proteins including EGF, FGF-2, IGF-1, NGF-ß, TGF-ß, VEGF, insulin, ß-catenin, caspase-3, caspase-8, Bcl-2, and Bcl-xL, which are important in cellular differentiation, proliferation, signalling, and apoptosis. Seven ?B crystallin sequences had strong interactions with FGF-2, NGF-ß, VEGF, insulin, and ß-catenin and mapped to the ß3-ß8-ß9 interface and surface domains in the N- and C-termini. This is the first report in which interactive sequences for regulatory proteins were identified in a sHSP. The remaining seven proteins did not interact with ?B crystallin. The ?B crystallin interactive sequences for regulatory proteins overlap with sequences for complex assembly, chaperone activity, and filament stabilization. The position of the interactive sequences in the structure of the ?B crystallin complex is consistent with a dynamic functional mechanism to coordinate exposure of interactive surfaces on ?B crystallin for the recognition of target proteins. Taken together, the results suggest the activity of sHSPs includes the regulation of key components of cellular differentiation, proliferation, signalling, and apoptotic pathways in stress and non-stress conditions.