Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and inhibits ER-stress induced apoptosis

Background The Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function. Methods Bag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni’s Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses. Results Our results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death. Conclusions We have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel interaction partner of Bag5.BioMed Central open acces

Selbstregulative Merkmale und die gesundheitsbezogene Lebensqualität von Jugendlichen während der COVID-19-Pandemie

Während der COVID-19-Pandemie wurde durch vermehrtes Stresserleben das subjektive Wohlbefinden von Jugendlichen auf die Probe gestellt. In Deutschland wurde der Unterricht von einem auf den anderen Tag auf Online-Formate umgestellt, soziale Kontakte wurden eingeschränkt und es war für die Jugendlichen schwierig bis unmöglich Freizeitaktivitäten nachzugehen. Unsere Studie untersuchte den Zusammenhang zwischen selbstregulativen Merkmalen – Selbstkontrolle (die Fähigkeit, innere Reaktionen zu verändern oder zu überwinden, unerwünschte Handlungstendenzen zu unterbrechen und Gefühle zu regulieren) und Need for Cognition (NFC; die intrinsische Motivation, sich mit kognitiv herausfordernden Aufgaben zu beschäftigen und Freude daran zu empfinden) – und der gesundheitsbezogenen Lebensqualität (HRQoL; Health-Related Quality of Life) bei belasteten Teenagern. Befunde mit erwachsenen Proband*innen zeigen Zusammenhänge zwischen selbstregulativen Merkmalen und subjektivem Wohlbefinden. Konkret geht eine höhere Selbstkontrolle mit einer besseren psychischen und physischen Gesundheit und einer stärkeren Emotionsregulierung sowie eine höhere NFC-Ausprägung mit mehr Lebenszufriedenheit und einem stärker ausgeprägten Selbstwertgefühl einher. Wir haben Fragebogendaten von insgesamt 311 Jugendlichen im Alter von 12 bis 18 Jahren, die mit verschiedenen Belastungen konfrontiert waren, erhoben. Jugendliche aus zwei Substichproben wiesen jeweils zusätzliche Belastungen wie eine chronische Erkrankung oder psychische Probleme auf. In Übereinstimmung mit bereits vorliegenden Ergebnissen für Erwachsene hingen auch in der untersuchten Stichprobe Selbstkontrolle und HRQoL eng positiv zusammen (r = .49). Außerdem sagte die NFC-Ausprägung als eigenständiger Prädiktor die HRQoL über Selbstkontrolle hinaus vorher. Unsere Ergebnisse zeigen einmal mehr, dass die NFC-Ausprägung, neben der Selbstkontrolle, eine wichtige Ressource für die Bewältigung von Herausforderungen im Leben darstellt. Mögliche Erklärungsansätze und detaillierte Analysen der Teilstichproben werden präsentiert

NFC as a resource for everyday challenges in school

Previous research results demonstrate that the investment trait Need for Cognition (NFC), i.e., the tendency to actively seek, engage in and enjoy effortful cognitive activity, is beneficial for learning behavior and consequently associated with better results in school and university. In our study we were particularly interested in the aspect of enjoying effortful cognitive activity. Current research demonstrates a relation between the joy of thinking and well-being as well as coping with not only intellectually demanding situations for students but also with challenges in everyday life. As everyday school life goes along with many challenges, we investigated how NFC could serve as a resource for general and school-specific well-being in children between 6 and 19 years. We collected survey data from 1538 children in central eastern Germany, stemming from eleven different schools spanning the school types elementary school, secondary school, grammar school and a school for intellectually highly gifted children. To account for the effects of variables already empirically proven to be influential, achievement motivation as well as academic self-concept were considered, too. In a mini meta-analysis we found a weighted mean of r = .41 between NFC and well-being in school highlighting the potential of NFC as a resource for coping with demanding situations. Hierarchical regression analyses showed varying degrees of incremental validity for NFC over and above achievement motives and ability self-concept depending on the type of school and variables considered. In primary school about 10% incremental variance explanation was due to NFC, but at secondary level only 1% additional variance could be explained by NFC

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

<div><p>The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in <em>in vivo</em> xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.</p> </div

The Bag-1 peptide inhibits prostate cancer cell growth <i>in vivo</i>.

<p>A. Stable clones of 22Rv.1 cells show reduced cell viability <i>in vitro</i>. Stable clones were seeded in a 96-well plate and the cell number in each well was estimated by measuring the ratio between the wavelength of excitation and emission 560/590 nm using the CellTiter-Blue™ proliferation assay. The bar charts represent the mean of at least four independent experiments ±SD (*p<0.05). B. Determination of the level of the Bag-1 expressed in the 22Rv.1 clones. Cellular extracts of 22v.1 stable clones were subjected to Western blot analysis. An anti-HA antibody was used to detect the peptide and an anti-β-actin antibody for equal loading control. C-D. The Bag-1 peptide reduces 22Rv.1 prostate tumor cell growth <i>in vivo</i>. Six-week old athymic nude mice were injected subcutaneously in both flanks with 5×10⁶ cells of each stable clone. Tumor size was measured once per week using a caliper and expressed as tumor volume in mm³. Shown are (C) the tumor volumes of clones transfected with empty expression vector and (D) clones expressing the Bag-1 peptide. Each point represents the mean volume and standard deviation of at least 5 to 10 tumors. E. Xenografts of stable clones expressing the Bag-1 peptide show increased apoptosis. Five-micron sections of formalin-fixed, paraffin-embedded tumor tissues were subjected to immunohistochemistry. Nuclei are stained blue-purple with hematoxylin while apoptotic cells detected with the TUNEL assay are stained brown. Representative sections of xenografts obtained from each 22Rv.1 stable clone were acquired with an Axioscop microscope (Zeiss). V: stands for empty vector expressing clone and P: peptide expressing clone.</p

Identification of Bag-1 peptide interfering with the action of GRP78/BiP.

<p>A. Diagrammatic representation of Bag-1 and its deletion mutants used for the identification of sequences required for binding the SBD of GRP78/BiP. Depicted in blue and red are the ubiquitin-like domain and the BAG domain respectively. B. Identification of the Bag-1 peptide binding to GRP78/BiP. GST-pull down assay performed incubating 500 µg lysates from HEK-293 cells transfected with the indicated Bag-1 constructs and 10 µg of GST-GRP78(SBD) or GST as control. Western blot analysis with an HA antibody to detected the Bag-1 mutants is shown. Coomassie blue staining was performed to demonstrate equal loading of the GST fusion proteins. C. The Bag-1 68 amino acid peptide binds <i>in vivo</i> to GRP78. HEK 293 cells were co-transfected with pcDNA-HA-tagged Bag-1 peptide. The transfected cells were treated with 2 mM Dithiobis(succinimidylpropionate) to cross-link the proteins and the cell lysates were immunoprecipitated with an anti-HA antibody or IgG as control, followed by Western blotting with an anti-GRP78/BiP antibody. D. The Bag-1 68 amino acid peptide inhibits GRP78/BiP refolding activity. <i>In vivo</i> luciferase refolding assay was performed in HEK-293 cells transfected at 70% confluency with 2 µg β-actin-fire fly luciferase construct, 3 µg pcDNA3 empty vector as control or pCMV6-GRP78/Bip, 6 µg of the indicated pcDNA3.1-Bag-1 or mutant constructs and 0.5 µg Renilla luciferase construct to determine the transfection efficiency. The transfected cells were divided into two. One half was left untreated and the other half was heat shocked at 45°C for 30 min. Thereafter luciferase activity was measured. Results are expressed as percentage of refolding activity relative to non-heat-shocked cells and presented as bar charts of the average of three independent experiments ± SEM. *p<0.05.</p

Effect of the Bag-1 peptide on tumor weights in 22Rv.1 and LNCaP xenograft tumor mouse models.

<p>Tumor weights and time of euthanasia of mice are recorded. The values represent the mean weights and standard deviation calculated from 4–10 tumors and the time of euthanasia.</p

An N-terminal unfolded fragment of the Bag-1 peptide is sufficient to reduce tumor cell growth.

<p>A. Schematic diagram of the Bag-1 peptide and its deletion mutants. The ubiquitin-like domain is represented in blue and the BAG domain in red. The numbers refer to the amino acid positions of the different domains. The amino acid residues are numbered following the numbering for Bag-1L. B. Prediction of the secondary structure of the Bag-1 peptide, showing an N-terminal β-hairpin from the ubiquitin-like domain (blue) and a C-terminal α-helix from the BAG domain (red). C. Normalized circular dichroism spectra of the Bag-1 peptides. 30 µM of the Bag-1 peptide were measured in 20 mM KHPO₄ buffer, pH 6.8 (black line). Its α-helical content was estimated to be approximately 25% by deconvolution of the spectra (red line). 12 µM of the N-term peptide (green line) and 11 µM of C-term (blue line) were measured under the same conditions. D. ¹H¹⁵N-HSQC NMR spectrum of ¹⁵N-labeled Bag-1 peptide (202–269) in 20 mm KHPO₄ buffer, pH 6.8, at 23°C. The narrow spectral dispersion indicates that the peptide does not exhibit a folded globular structure. The H_δ and H_ε side chain signals of asparagine and glutamine are connected by thin lines. E. The N-terminal region of the Bag-1 peptide is important for GRP78 binding. 400 µg of 22Rv.1 cell lysate were incubated with glutathione-agarose beads carrying 15 µg GST-N-term peptide (lane 2), GST-C-term peptide (lane 3), GST-ΔUbi peptide (lane 4), GST-Bag-1(202–269) peptide (lane 5) and GST (lane 6). The beads were washed and the bound proteins were separated by 10% SDS-PAGE and subjected to Western blotting using antibodies directed against GRP78 or GST. The input lane shows 1/10 aliquot of cell lysate used for the study. F. Clonogenic assay of the Bag-1 peptides expressed in 22Rv.1 cells. Cells transfected with the indicated constructs were selected in medium containing neomycin and the colonies formed were quantified. Shown are the mean value ±SEM of at least three independent experiments using three different plasmid preparations (*p<0.05). G-I. The N-terminal peptide reduces tumor growth <i>in vivo</i>. Six-week old athymic nude mice were injected subcutaneously on both flanks with 5×10⁶ cells of each stable clone. Tumor size was measured once per week using a caliper and expressed as tumor volume in mm³. Shown are the tumor volumes of clones transfected with the N-terminal peptide (G), the C-terminal peptide (H) and the ΔUbi peptide (I). Each point represents the mean volume and standard deviation of at least 5 to 10 tumors.</p