Stress-70 Protein Research Articles

Acetylation of mtHSP70 at Lys595/653 affecting its interaction between GrpEL1 regulates glioblastoma progression via UPRmt

BackgroundThe mitochondrial unfolded protein response (UPRmt) is a vital biological process that regulates mitochondrial protein homeostasis and enables glioblastoma cells to cope with mitochondrial oxidative stress in the tumor microenvironment. We previously reported that the binding of mitochondrial stress-70 protein (mtHSP70) to GrpE protein homolog 1 (GrpEL1) is involved in the regulation of the UPRmt. However, the mechanisms regulating their binding remain unclear. Herein, we examined the UPRmt in glioblastoma and explored whether modulating the interaction between mtHSP70 and GrpEL1 affects the UPRmt. MethodsWestern blot analysis, aggresome staining, and transmission electron microscopy were used to detect the activation of the UPRmt and protein aggregates within mitochondria. Molecular dynamics simulations were performed to investigate the impact of different mutations in mtHSP70 on its binding to GrpEL1. Endogenous site-specific mutations were introduced into mtHSP70 in glioblastoma cells using CRISPR/Cas9. In vitro and in vivo experiments were conducted to assess mitochondrial function and glioblastoma progression. ResultsThe UPRmt was activated in glioblastoma cells in response to oxidative stress. mtHSP70 regulated mitochondrial protein homeostasis by facilitating UPRmt-progress protein import into the mitochondria. Acetylation of mtHSP70 at Lys595/653 enhanced its binding to GrpEL1. Missense mutations at Lys595/653 increased mitochondrial protein aggregates and inhibited glioblastoma progression in vitro and in vivo. ConclusionsWe identified an innovative mechanism in glioblastoma progression by which acetylation of mtHSP70 at Lys595/653 influences its interaction with GrpEL1 to regulate the UPRmt. Mutations at Lys595/653 in mtHSP70 could potentially serve as therapeutic targets and prognostic indicators of glioblastoma.

Pathological Conversion of Mouse Perivascular Adipose Tissue by Notch Activation.

Perivascular adipose tissue (PVAT) surrounding arteries supports healthy vascular function. During obesity, PVAT loses its vasoprotective effect. We study pathological conversion of PVAT, which involves molecular changes in protein profiles and functional changes in adipocytes. Approach and Results: C57BL6/J mice were fed a 60% high-fat diet for 12 weeks or a cardioprotective 30% calorie-restricted diet for 5 weeks. Proteomic analysis identified PVAT as a molecularly distinct adipose depot, and novel markers for thermogenic adipocytes, such as GRP75 (stress-70 protein, mitochondrial), were identified. High-fat diet increased the similarity of protein signatures in PVAT and brown adipose, suggesting activation of a conserved whitening pathway. The whitening phenotype was characterized by suppression of UCP1 (uncoupling protein 1) and increased lipid deposition, leptin, and inflammation, and specifically in PVAT, elevated Notch signaling. Conversely, PVAT from calorie-restricted mice had decreased Notch signaling and less lipid. Using the Adipoq-Cre strain, we constitutively activated Notch1 signaling in adipocytes, which phenocopied the changes in PVAT caused by a high-fat diet, even on a standard diet. Preadipocytes from mouse PVAT expressed Sca1, CD140a, Notch1, and Notch2, but not CD105, showing differences compared with preadipocytes from other depots. Inhibition of Notch signaling during differentiation of PVAT-derived preadipocytes reduced lipid deposition and adipocyte marker expression. PVAT shares features with other adipose depots, but has a unique protein signature that is regulated by dietary stress. Increased Notch signaling in PVAT is sufficient to initiate the pathological conversion of PVAT by promoting adipogenesis and lipid accumulation and may thus prime the microenvironment for vascular disease.

Expression of HSP70 family mRNAs in albino northern snakehead, Channa argus: Response to extreme temperature stress and bacterial infection

The wild albino northern snakehead, Channa argus, has only been found in Jialing Rivers System of China so far. It is easy to be affected by the environmental factors such as temperature changes and bacterial infection, thus causing a huge economic loss. Therefore, this study cloned a 2,213 bp cDNA that encodes a protein of heat shock protein 70 (CaHSP70), which has an open reading frame (ORF) that encodes 639 amino acids and the corresponding polypeptides of 70.50 kDa. And the oretical isoelectric point (pI) of CaHSP70 is 5.79. Additionally, we also cloned a cDNA for heat shock cognate protein 70 (CaHSC70) with a total length of 2,300 bp. And the ORF of CaHSC70 encodes 648 amino acids and 71.18 kDa peptides. The pI of CaHSC70 is 5.22. Moreover, the cDNA length of stress-70 protein mitochondrial (CaHSPA9) is 2,944 bp with an ORF that encodes 679 amino acids, polypeptides of 73.74 kDa, and a pI of 6.68. The quantitative real-time PCR (qRT-PCR) analysis showed that the mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9 genes were tissue-specific in the control groups. After the heat shock at 37 °C, the mRNA expression levels of CaHSP70 were extremely significantly upregulated in the kidney, liver, spleen, and brain tissues, while fewer mRNA expression levels of CaHSC70 and CaHSPA9 showed a relatively induction in these tissues. In contrast, after the cold shock at 8.5 °C, fewer mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9 showed the changes of expression in all the tissues, compared to heat shock. In addition, CaHSP70, CaHSC70, and CaHSPA9 mRNA expression levels showed an overall trend of first upregulating and then falling after Edwardsiella tarda (strain No. DL1,476) challenge. In conclusion, this study demonstrated that temperature had a great effect on the mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9, and the mRNA expression levels of these three genes were also sensitive to pathogen infection, especially CaHSP70 in the albino C. argus.

Mass spectrometry and DigiWest technology emphasize protein acetylation profile from Quisinostat-treated HuT78 CTCL cell line

Histone deacetylases (HDACs) are key enzymes involved in epigenetic modulation and were targeted by HDAC inhibitors (HDACis) for cancer treatment. The action of HDACis is not restricted to histones and also prevents deacetylation of other proteins, supporting their wide biological actions. The HuT78 cell line is recognized as a key tool to support and understand cutaneous T-cell lymphoma (CTCL) biology and was used as a predictive model since HDACi such as Vorinostat and Panobinostat have both demonstrated apoptotic activities in HuT78 cells and in primary blood CTCL cells. In this study, Quisinostat (JNJ-26481585) a novel second-generation HDACi with highest potency for HDAC1, was tested on HuT78 cell line. Quantitative mass spectrometry (MS)-based proteomics after acetylated-lysine peptide enrichment and a targeted antibody-based immunoassay (DigiWest) were used as complementary technologies to assess the modifications of the acetylated proteome. As expected, several acetylated lysines of histones were increased by the HDACi. Additional acetylated non-histone proteins were modulated after treatment with Quisinostat including the nucleolin (a major nucleolar protein), the replication protein A 70 kDa DNA-binding subunit, the phosphoglycerate kinase 1, the stress-70 protein, the proto-oncogene Myc and the serine hydroxymethyltransferase. A better knowledge of histone and non-histone acetylated protein profile after Quisinostat treatment can strongly support the understanding of non-clinical and clinical results of this HDACi. These technological tools can also help in designing new HDACis in a pharmaceutical drug discovery program. SignificanceA better knowledge of histone and non-histone acetylated protein profile after HDAC inhibitors (HDACis) treatment can strongly support the understanding of non-clinical and clinical investigations in a pharmaceutical drug discovery program. Relative quantification using mass spectrometry -based proteomics after acetylated-lysine peptide enrichment and a targeted antibody-based immunoassay (DigiWest) are proposed as complementary technologies to assess the modifications of the acetylated proteome. Quisinostat (JNJ-26481585) a novel second-generation HDACi with highest potency for HDAC1 was better characterized in vitro in HuT78 cells to support and understand cutaneous T-cell lymphoma (CTCL) therapeutic research program.

Proteomic Profiling of Liver and Plasma in Chronic Ethanol Feeding Model of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice.

Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH- ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). ADH- deer mice were fed 3.5g% EtOH via Lieber-DeCarli liquid diet daily for 3months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10-3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. Chronic EtOH feeding in ADH- deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid β oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate metabolism, in cell regulation and architecture. Reduced expression of various carrier proteins as found in the plasma of EtOH group has a biomarker potential.

Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins.

Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer.

Comparative proteomic analysis of paclitaxel resistance-related proteins in human breast cancer cell lines.

Paclitaxel is widely used to treat various cancers; however, resistance to this drug is a major obstacle to breast cancer chemotherapy. To identify the proteins involved in paclitaxel resistance, the present study compared the proteomes of MCF-7 human breast cancer cells and its paclitaxel-resistant subclone MCF-7/PTX. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry, 11 upregulated and 12 downregulated proteins were identified in MCF-7/PTX cells compared with the parental cell line. These 23 proteins were functionally classified as stress-induced chaperones, metabolic enzymes and cytoskeletal proteins. The anti-apoptotic proteins, stress-70 protein, 78-kD glucose-regulated protein, peptidyl-prolyl cis-trans isomerase A (PPIA) and heterogeneous nuclear ribonucleoprotein H3, were also upregulated in MCF-7/PTX cells. Notably, knockdown of the stress-response chaperone PPIA using small interfering RNA in MCF-7/PTX cells restored their sensitivity to paclitaxel. These findings indicated that PPIA may have an important role in paclitaxel resistance in MCF-7/PTX cells.

Ischemic postconditioning influences electron transport chain protein turnover in Langendorff-perfused rat hearts.

Ischemia postconditioning (IPo) is a promising strategy in reducing myocardial ischemia reperfusion (I/R) injury (MIRI), but its specific molecular mechanism is incompletely understood. Langendorff-perfused isolated rat hearts were subjected to global I/R and received IPo in the absence or presence of the mitochondrial ATP-sensitive potassium channel (mitoKATP) blocker 5-hydroxydecanoate (5-HD). Myocardial mitochondria were extracted and mitochondrial comparative proteomics was analyzed. IPo significantly reduces post-ischemic myocardial infarction and improved cardiac function in I/R rat hearts, while 5-HD basically cancelled IPo’s myocardial protective effect. Joint application of two-dimensional polyacrylamide gel electrophoresis (2DE) and MALDI-TOF MS identified eight differentially expressed proteins between groups. Expression of cardiac succinate dehydrogenase (ubiquinone) flavoprotein subunit (SDHA) increased more than two-fold after I/R, while IPo led to overexpression of dihydrolipoyl dehydrogenase (DLD), NADH dehydrogenase (ubiquinone) flavoprotein 1 and isoform CRA_b (NDUFV1). When the mitoKATP was blocked, MICOS complex subunit Mic60 (IMMT) and Stress-70 protein (Grp75) were over expressed, while DLDH, ATPase subunit A (ATPA) and rCG44606 were decreased. Seven of the differential proteins belong to electron transport chain (ETC) or metabolism regulating proteins, and five of them were induced by closing mitoKATP in I/R hearts. We thus conclude that IPo’s myocardial protective effect relies on energy homeostasis regulation. DLD, SDHA, NDUFV1, Grp75, ATPA and rCG44606 may contribute to IPo’s cardial protective effect.

Abstract 1539: Analysis of differential protein profiles in co-culture models of pancreatic cancer cells and fibroblasts

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies. The vast majority of patients with advanced disease die within a year of diagnosis. Tumor microenvironment plays a critical role in tumor initiation, progression, metastasis, and response to treatment in PDAC. Pancreatic adenocarcinoma cells often appear to grow within a fibrotic, abundant, and poorly perfused stroma, with epithelial cells frequently accounting for <20% of the tumor volume. Cellular components of tumor stroma include fibroblasts, macrophages, and stellate cells plus extracellular proteins. Moreover, the network of tumor stroma imposes a barrier for drug delivery. The aim of this study was to search for novel protein biomarkers of tumor-stroma interaction for a better understanding and intervention of PDAC. Methods: 1- Co-cultures between two pancreatic adenocarcinoma cell lines (PL45 and Capan-1) and embryonic fibroblasts (LC5) were established. LC5 cell line was stably transfected with Green Fluorescent Protein (GFP) to allow their detection, and later separation by flow cytometry, of the co-cultures. 2- In addition, tumor cells were grown with fibroblasts secretome. 3- After 72 hours of co-culture, tumor cells were isolated and protein extraction was performed. 4- Proteins differentially expressed in tumor cells grown in co-cultures, or in tumor cells grown with fibroblasts secretome, versus in tumor cells monocultures, were identified by using two-dimensional electrophoresis-based proteomic tools (2D-DIGE) followed by mass spectrometry (MS/MS) of the spots of interest. Results: A total of 105 differentially expressed proteins were documented between PL45 cells growing in mono-culture, co-culture and conditioned medium. Also, 161 differentially expressed proteins were recognized under similar conditions in the Capan1 cell line. Our study identified seventeen different proteins mainly involved in cytoskeleton organization (Filamin B, Alpha-actinin-4, Annexin A2, Radixin, T-complex protein 1 subunit epsilon, Actin cytoplasmic 1); regulation of cell migration and adhesion (Prelamin-A/C, 40S ribosomal protein SA); protein biosynthesis (EF-2); DNA damage and repair response (VCP, XRCC6); nucleotide biosynthetic process (MTHFD1) and, heat-stress response (HSPD1, GRP78, STIP1, CCT5, Stress-70 protein mitochondrial). Conclusion: This in vitro model identifies several processes that might be responsible for the tumor-stromal interactions occurring in vivo, which should be considered potential targets for therapeutic interventions in PDAC, in addition to those targets already known to exist in PDAC cells. Citation Format: Elena Prieto-García, M. Teresa Agulló-Ortuño, C. Vanesa Díaz-García, Inés García-Consuegra, Jorge Adeva, M. Carmen Riesco, Lucía Parrilla, Carlos Gómez, Laura Lema, Luis Robles, Hernán Cortés-Funes, José A. López-Martín. Analysis of differential protein profiles in co-culture models of pancreatic cancer cells and fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1539. doi:10.1158/1538-7445.AM2015-1539

A proteomic analysis on human sperm tail: comparison between normozoospermia and asthenozoospermia.

Asthenozoospermia is a common cause of human male infertility characterized by reduced sperm motility. The molecular mechanism that impairs sperm motility is not fully understood. This study proposed to identify novel biomarkers by focusing on sperm tail proteomic analysis of asthenozoospermic patients. Sperm were isolated from normozoospermic and asthenozoospermic semen samples. Tail fractions were obtained by sonication followed by Percoll gradient. The proteins were extracted by solubilization and subjected to two-dimensional gel electrophoresis (2-DE); then, the spots were analyzed using Image Master 2D Platinum software. The significantly increased/decreased amounts of proteins in the two groups were exploited by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI-TOF-TOF) mass spectrometry. Three hundred ninety protein spots were detected in both groups. Twenty-one protein spots that had significantly altered amounts (p < 0.05) were excised and exploited using MALDI-TOF-TOF mass spectrometry. They led to the identification of the following 14 unique proteins: Tubulin beta 2B; glutathione S-transferase Mu 3; keratin, type II cytoskeletal 1; outer dense fiber protein 2; voltage-dependent anion-selective channel protein 2; A-kinase anchor protein 4; cytochrome c oxidase subunit 6B; sperm protein associated with the nucleus on the X chromosome B; phospholipid hydroperoxide glutathione peroxidase-mitochondrial; isoaspartyl peptidase/L-asparaginase; heat shock-related 70kDa protein 2; stress-70 protein, mitochondrial; glyceraldehyde-3-phosphate dehydrogenase, testis-specific and clusterin. Fourteen proteins present in different amounts in asthenozoospermic sperm tail samples were identified, four of which are reported here for the first time. These proteins might be used as markers for the better diagnosis of sperm dysfunctions, targets for male contraceptive development, and to predict embryo quality.

Abstract 4998: In-depth analysis of myoma organotypic model can determine potential markers of oral cancer invasion

Abstract Oral squamous cell carcinoma (OSCC) is the eighth most prevalent malignant neoplasm and accounts for 2% of all deaths by cancer worldwide. OSCCs have a highly variable clinical course, and because it is often diagnosed only after it has reached an advanced stage, the overall survival rate is less than 50% in 5 years. A better knowledge of the mechanisms that influence OSCC development and its progression is still needed. Particularly, most of the attention has been driven to mechanisms associated with tumor cell invasion and how the surrounding tumoral stroma may influence it. The myoma organotypic model is a novel fully human organotypic invasion model that mimics the tumor microenvironment (TME) (Nurmenniemi et al. 2009). Aiming to investigate the molecular mechanisms related to OSCC development and search for prognostic markers for this tumor, we used the myoma organotypic assay, followed by MS-ESI technique of laser microdissected invaded tissue, to analyze several features related to OSCC invasive vs. non-invasive cells. Our results showed that the HSC-3, an aggressive and invasive oral squamous cell carcinoma cell line, invaded as small islands and cell chords and penetrated deeply in the tissue. Unlike, the less aggressive LN2 cells, formed a well structured epithelium layer, but they invaded less in myoma. The proteomic analysis was able to characterize the differential expressed proteins of these, and revealed a group of proteins exclusively expressed in the cells that deeply invade the myoma, such as HMGA2 (high mobility group), JUP (junction plakoglobin), PLEC (plectin), HIST1H2B (Histone H2B), IQGAP1 (Ras GTPase-activating-like protein), EEF1A1 (elongation factor 1-alpha), HSPA9 (Stress-70 protein, mitochondrial), HADHA (Trifunctional enzyme subunit alpha). Additionally, the TME is a unique ambient created and shaped by the tumor, which orchestrates molecular and cellular events taking place in surrounding tissues. Interestingly, we found a group of high abundant proteins expressed in the TME surrounding the invasive island. Among them are PALLD (palladin), TNC (tenascin C), LMNA (laminin), VIM (vimentin), and COL1A (collagen-type I-alpha I), which have been described to be involved in cell migration and proliferation. Our preliminary data suggests that the myoma organotypic tumor invasion assay is successful in determining new molecules and potential pathways associated with OSCC tumor invasion. We also present here a new insight for head and neck cancer biomarkers research that can, ultimately, contribute to more individualized treatment of patients affected by OSCC. However, validation of such results in clinical sample cohorts is important for a better understanding of the biological events associate with tumor invasion and disease spread (metastasis) in those patients. Citation Format: Nilva K. Cervigne, Carolina Bitu, Bianca A. Pauletti, Adriana F. Paes Leme, Tuula Salo, Ricardo Della Coletta. In-depth analysis of myoma organotypic model can determine potential markers of oral cancer invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2014-4998

Proteomic Analysis of Celecoxib on Chondrocytes from Patients with Osteoarthritis

Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chondrocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins were extracted from the cells and subjected to 2-dimensional differential image gel electrophoresis (2D-DIGE). Proteins of interest were identified by mass spectrometry. Results: Eighty-six protein spots showed significantly different intensities with each reagent or reagent combination. AAA+ protein, HSP47/Serpin, cAMP-dependent protein kinase type II-beta regulatory subunit, alpha-actin-4 and tubulin decreased with the addition of celecoxib, while apolipoprotein A-V, glutamate carboxipeptide 2, mitochondrial stress-70 protein, sorting nexin-9 and GRP78 increased with the addition of celecoxib. GRP78 is a stress protein and may be chondroprotective. Celecoxib modulated IL-1β stimulated chondrocytes, and CD200R and moesin were identified as such resulting proteins. Conclusion: Protein profiles of OA chondrocytes changed after administration of celecoxib. Further investigation is needed to elucidate the function of each protein in OA chondrocytes.

Identification of Mitochondrial Proteins regulated during Activation of GPER1-Leading to Cardioprotection

Several studies have demonstrated that G-protein coupled estrogen receptor (GPER1) can directly bind to estrogen and mediate its action. We previously demonstrated that GPER1 activation with G1, a specific agonist is cardioprotective in wild-type (WT) mice subjected to ischemia/reperfusion injury. Here, we identified regulated mitochondrial proteins associated with GPER1 activation by estrogen treatment in WT and GPER1-/- mouse hearts after ischemia/reperfusion. Isolated hearts from male WT (C57BL/6NCrL) or GPER1-/- mice were perfused using the Langendorff technique with Krebs Henseleit buffer, with and without (control) the addition of estrogen (40 nM). Hearts were subjected to 18 min global ischemia followed by 10min reperfusion. Mitochondria were isolated, and 2D-DIGE followed by mass spectrometry was performed. Proteins of interest were the ones (up- or down- regulated) in WT+E2 vs. WT-control that remained unchanged in GPER-/-+estrogen vs. GPER-/--control and WT-control vs. GPER-/--control. Robust changes of proteins were observed in 45 spots, out of which 14 were down regulated and 31 up regulated. In these 45 spots, 52 unique proteins were identified. Among the proteins identified, estrogen treatment induced the regulation of enzymes mostly involved in electron transfer chain and ATP production. Estrogen action involved the down-regulation of filament proteins (filamin A,B,C), the up-regulation of proteins that activate transcription, and proteins involved in the contractile system (tropomyosin and myosin). Further, estrogen treatment is associated with regulation of proteins acting in stress (stress-70 protein, and 60 kDa heat shock protein), in cell communication (glial fibrillary acidic protein), and in signaling pathways (membrane-associated phosphatidylinositol transfer protein). Finally, estrogen down-regulated the mitochondrial inner membrane protein, this protein controls mitochondrial cristae morphology. In conclusion, rapid estrogen effects through GPER1 activation are associated with the down- or up-regulation of mitochondrial and non-mitochondrial proteins likely in close contact to mitochondria.

Low Dose-Radiation of Bone Marrow Stromal Cells Supports Pre-Leukemic Cell Survival Via Adhesion and Inflammatory Signaling Stimulation

Abstract 4610Low-dose irradiation (LDI) exposure is a form of environmental carcinogen, which is of significant interest after the nuclear accident in Japan. In this study, we investigated the LDI-induced molecular alterations of bone marrow (BM) –derived stromal cells (MSCs), and the biological response of pre-leukemic cells neighbor to LDI exposed MSCs. As the model of pre-leukemic cells, we utilized the Epstein- Barr virus (EBV) infected and immortalized B lymphocyte cell line (EBV-B).In MSCs, exposed to 100 mGy irradiation (4MVX rayfrom a LINAC) caused cell growth inhibition (75.8±2.4 % of control, p<0.001, MTT assay) with moderate apoptosis induction (SubG1 %; control 2.3±0.4, radiation 7.3±2.3, p=0.03, PI cell cycle analysis) at 24 hrs. Screening up to 28,869 genes by cDNA microarray (Human Gene 1.0 ST Array, Affymetrix) revealed 48 up-regulated and 45 down-regulated genes (>1.3 fold) in irradiated MSCs (at 24 hrs) compared to non-irradiated MSCs. The functional network analysis of cDNA microarray data by MetaCore (GeneGo) showed an enrichment of the adhesion/ECM remodeling pathways. TaqMan RT-PCR confirmed significant up-regulation of ECM scaffold Sulfatase1 mRNA (2.0±0.1 fold, p=0.004). Proteomic analysis utilized isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems), identified 32 up-regulated and 1 down-regulated proteins (p<0.05) among 1,536 detected proteins comparing irradiated MSCs (at 24 hrs) with non-irradiated MSCs. KEGGontology analysis (Kyoto University) found the activation of focal adhesion and apoptosis pathways in irradiated MSCs; among 32 up-regulated proteins, six are involoved in adhesion/cytoskeleton regulation (Prelamin-A; p<0.001, Catenin beta-1; p=0.03, Keratin type II; p=0.02, Collagen alpha1; p=0.02, alpha actinin1 and 4; p=0.01, p=0.02) and three are involved in apoptosis/senescence (Stress-70 protein l; p=0.02, Endoplasmin; p=0.008, Prohibitin; p=0.04). These changes were accompanied with increased secretion of inflammation and coagulation marker protein plasminogen activator inhibitor 1 (PAI-1) from irradiated MSCs (control 10.1±0.03 ng/mL, radiation >15.0 ng/mL, at 72hrs). We then investigated the effect of LDI exposed MSCs on EBV-B cell proliferation. Under low-serum growth condition (1% FBS), co-culture with pre-irradiated (100 mGy) MSC layers supported EBV-B cell proliferation more prominently than non-irradiated MSCs (118.3±4.0 % viable cell number, p<0.001, 72hrs). Gene expression profiles by cDNA microarray and sequenced MetaCore ontology revealed 53 up-regulated and 39 down-regulated genes showing an enrichment of the coagulation and cell adhesion pathways in EBV-B cells co-cultured with pre-irradiation of MSCs compared to the ones co-cultured with non-irradiated MSCs. Up-regulation of anti-apoptotic BCL2 mRNA (0.5±0.03 fold, p=0.02) and inflammatory IL8 mRNA (2.0±0.03 fold, p<0.001) was further detected by TaqMan RT-PCR. The purity of EBV-B cells separated from MSCs was confirmed by lack of CD90 mRNA expression by PCR. These data suggest that LDI exposure to MSCs leads to latent adhesion and inflammatory signaling in MSCs, which potentially activates pro-survival pathways of co-cultured pre-leukemic EBV-B cells. In summary, we present the biosignature of the response of BM-derived stromal cells to LDI utilizing gene array and quantitative proteomics analysis that may have important implications for risk assessment in environmental medicine. Disclosures:No relevant conflicts of interest to declare.

Functional specific roles of H‐ras and N‐ras. A proteomic approach using knockout cell lines

Ras small GTPases function as transducers of extracellular signals regulating cell survival, growth and differentiation. There are three major ras isoforms: H-, N- and K-Ras. To improve the understanding of H- and N-Ras protein signalling networks, we compared total proteome changes in mouse embryonic fibroblasts knock out for H-ras and/or N-ras, using proteomics tools combining 2DE, semi-quantitative image analysis, in-gel trypsin digestion and mass spectrometry. There are four up-regulated proteins due to the loss of expression of H-Ras (including cyclin-dependent kinase inhibitor 2A) and eight down-regulated (including stress-70 protein, dihydropyrimidinase-related-protein 3, heat shock cognate 71 kDa protein, tropomyosin beta chain, Rho GDP-dissociation inhibitor 1) and six up-regulated proteins (e.g. leukocyte elastase inhibitor A, L-lactate dehydrogenase B chain, c-Myc-responsive protein Rcl, interleukin-1 receptor antagonist protein) due to the loss of expression of both N- and H-Ras. Most of these proteins are related to Ras signalling in one way or another. Changes in expression of some of these proteins were further confirmed by Western blot. This proteomic comparative analysis from loss of function of H- and N-Ras knockout fibroblasts yields interpretable data to elucidate the differential protein expression, and contributes to evaluate the possibilities for physiological and therapeutic targets.

The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry

Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4-5 and 5-6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development.

2D-PAGE of ovarian cancer: Analysis of soluble and insoluble fractions using medium-range immobilized pH gradients

Ovarian cancer remains a leading cause of cancer death. A comparative proteomic study was performed on normal ovarian tissue ( n = 5) and grade 3 ovarian tumours ( n = 5) to search for differentially expressed proteins. In contrast to other studies, here we extracted proteins in soluble and insoluble protein fractions using commercial kits and also utilised three medium-range IPG strips that encompassed the broad pH range of 3–10 (pH 3–6, 5–8 and 7–10). Protein fractions were compared by 2D-PAGE and MALDI-TOF/TOF-MS. Nineteen differentially expressed proteins were identified: HSP60, Grp78, CK19, EF-Tu, MRLC2, prohibitin, Stress-70 protein, TPI and tubulin α6 were up-regulated in grade 3 tumours whereas annexin A2 and A5, antithrombin-III precursor, CBR1, GSTM2, GSTM3, RALDH1, serum albumin precursor, transthyretin precursor and vimentin were found to be down-regulated in grade 3 ovarian tumours. These proteins are associated with cytoskeleton rearrangement, cell metabolism, tumour suppression function, apoptosis and induction of host response.

An assessment of drilling disturbance on Echinus acutus var. norvegicus based on in-situ observations and experiments using a remotely operated vehicle (ROV)

An industrial work-class remotely operated vehicle (ROV) carried out a video survey of epibenthic megafauna before and after physical disturbance caused by the drilling of a hydrocarbon exploration well in the North Sea, off Norway at 114 m water depth. Megafaunal density, in general, had declined one month after the drilling event. There were particular declines in the density of the numerically dominant echinoid, Echinus acutus var. norvegicus, in a 50 m zone around the drill site. The ROV was also used to carry out in-situ experiments to assay the effects of drilling disturbance (via sediment burial) on E. acutus and, in addition, to determine any consequence upon E. acutus of retrieval to the surface (due to decompression and temperature change). Changes in the expression of an inducible stress-70 protein in echinoid Aristotle's lantern muscle and intestine were evaluated using quantitative real-time PCR. Sediment burial caused a significant increase in the expression of a stress-70 gene from the echinoids' Aristotle's lantern muscle tissue. Repeated echinoid retrieval to the sea surface did not result in any significant alteration of stress-70 gene expression. These experiments show the utility of ROVs in linking ecological observations with real-time in-situ experimental manipulations, and that investigative gene expression techniques can be incorporated in the subsequent analysis of retrieved specimens.

Identification of osteocyte‐selective proteins

Since little is known regarding osteocytes, cells embedded within the mineralized bone matrix, a proteomics approach was used to discover proteins more highly expressed in osteocytes than in osteoblasts to determine osteocyte-specific function. Two proteomic profiles obtained by two different proteomic approaches using total cell lysates from the osteocyte cell line MLO-Y4 and the osteoblast cell line MC3T3 revealed unique differences. Three protein clusters, one related to glycolysis (Phosphoglycerate kinase 1, fructose-bisphosphate aldolase A, hypoxia up-regulated 1 [ORP150], triosephosphate isomerase), one to protein folding (Mitochondrial Stress-70 protein, ORP150, Endoplasmin), and one to actin cytoskeleton regulation (Macrophage-capping protein [CapG], destrin, forms of lamin A and vimentin) were identified. Higher protein expression of ORP-150, Cap G, and destrin in MLO-Y4 cells compared with MC3T3 cells was validated by gene expression, Western blotting, and in vivo expression. These proteins were shown to be selective in osteocytes in vivo using immuno-staining of mouse ulnae. Destrin was most highly expressed in embedding osteoid osteocytes, GapG in embedded osteocytes, and ORP150 in deeply embedded osteocytes. In summary, the proteomic approach has yielded important information regarding molecular mechanisms used by osteocytes for embedding in matrix, the formation of dendritic processes, and protection within a hypoxic environment.

Bone marrow mononuclear cells reduce myocardial reperfusion injury by activating the PI3K/Akt survival pathway

Objective Adult bone marrow mononuclear cells (BMMNCs) can restore cardiac function following myocardial necrosis. Protocols used to date have administered cells relatively late after ischaemia/reperfusion injury, but there is the opportunity with elective procedures to infuse cells shortly after restoration of blood flow, for example after angioplasty. Our aim was therefore to try and quantify protection from myocardial injury by early infusion of BMMNCs in a rat ischaemia reperfusion (I/R) model. Methods and results Male Wistar rats underwent 25 min of ischaemia followed by 2 h reperfusion of the left anterior descending coronary artery. Ten million BMMNCs were injected i.v. at reperfusion. We found BMMNCs caused a significant reduction in infarct size at 2 h when assessed by staining the area at risk with p-nitro blue tetrazolium (42% reduction, P < 0.01). Apoptosis and necrosis of isolated cardiomyocytes was significantly reduced in the area at risk. Functional assessment at 7 days using echocardiography and left ventricular catheterisation showed improved systolic and diastolic function in the BMMNC treatment group (LVEF: BMMNC 71 ± 3% vs. PBS 48 ± 4%, P < 0.0001). In functional studies BMMNC injected animals showed increased activation of Akt, inhibition of GSK-3β, amelioration of p38 MAP kinase phosphorylation and NF-κB activity compared to control myocardium. Inhibition of PI3K with LY294002 abolished all beneficial effects of BMMNC treatment. Proteomic analysis also demonstrated that BMMNC treatment induced alterations in proteins within known cardioprotective pathways, e.g., heat shock proteins, stress-70 protein as well as the chaperone protein 14-3-3 epsilon. Conclusions Early BMMNC injection during reperfusion preserves the myocardium, with evidence of reduced apoptosis, necrosis, and activation of survival pathways.