It has been shown that drug resistance is extremely common in hepatocellular carcinoma (HCC) and is one of the major problems in HCC chemotherapy. However, the detailed mechanisms remain largely unknown. We have previously shown that endoplasmic reticulum (ER) stress is involved in the tumorigenesis of HCC. Here, we demonstrated that the unfolded protein response (UPR) inhibits cisplatin-induced HCC cell apoptosis. In HCC cells, cisplatin treatment triggers the UPR, which subsequently inhibits cisplatin-induced apoptosis. Importantly, mild ER stress precondition suppresses the sensitivity of HCC cells to cisplatin-induced apoptosis through autophagy regulation. Furthermore, heat-shock protein 27 (Hsp27) is involved in the cytoprotective role of the UPR in cisplatin-induced apoptosis. We also demonstrated that Hsp27 inhibits cisplatin- induced HCC cell death through autophagy activation. Taken together, our results indicate that the UPR inhibits cisplatin-induced apoptosis in HCC cells, at least in part, by Hsp27-mediated autophagy activation.

• MeSH
• Apoptosis physiology   drug effects   MeSH
• Autophagy physiology   drug effects   MeSH
• Cisplatin pharmacology   metabolism   MeSH
• Dithiothreitol pharmacology   MeSH
• Endoplasmic Reticulum physiology   drug effects   MeSH
• Carcinoma, Hepatocellular pathology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Liver Neoplasms pathology   MeSH
• HSP27 Heat-Shock Proteins genetics   metabolism   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Heat-Shock Response MeSH
• Unfolded Protein Response physiology   MeSH
• Tunicamycin pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

AIMS: Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. METHODS: We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). RESULTS: We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. CONCLUSIONS: Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.

• MeSH
• alpha-Synuclein * metabolism   MeSH
• Biomarkers metabolism   MeSH
• Endoplasmic Reticulum Chaperone BiP * metabolism   MeSH
• Lewy Body Disease * pathology   metabolism   MeSH
• Eukaryotic Initiation Factor-2 * metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Brain metabolism   pathology   MeSH
• Nerve Growth Factors metabolism   MeSH
• Heat-Shock Proteins * metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Unfolded Protein Response * physiology   MeSH
• Endoplasmic Reticulum Stress physiology   MeSH
• Up-Regulation * MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

• MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• A549 Cells MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Phosphorylation genetics   physiology   MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Mutation genetics   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Polyribosomes metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   metabolism   MeSH
• Protein Stability MeSH
• Intrinsically Disordered Proteins genetics   metabolism   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BRAFV600E mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III BRAFMT (discovery set: n = 31; validation set: n = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the BRAFMT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the BRAFMT colorectal cancer subgroup with poorest outcome. We also found that oncogenic BRAF drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in BRAFMT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in BRAFMT in vitro and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic BRAF induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic BRAFMT colorectal cancer. Mol Cancer Ther; 17(6); 1280-90. ©2018 AACR.

• MeSH
• Apoptosis drug effects   genetics   MeSH
• Models, Biological MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Colorectal Neoplasms drug therapy   genetics   metabolism   mortality   MeSH
• Hydroxamic Acids pharmacology   MeSH
• Humans MeSH
• MAP Kinase Signaling System MeSH
• Mutation * MeSH
• Biomarkers, Tumor MeSH
• Cell Line, Tumor MeSH
• Oligopeptides pharmacology   MeSH
• Prognosis MeSH
• Heat-Shock Proteins genetics   metabolism   MeSH
• Protein Biosynthesis MeSH
• Antineoplastic Agents pharmacology   MeSH
• Proto-Oncogene Proteins B-raf antagonists & inhibitors   genetics   metabolism   MeSH
• Pyrimidines pharmacology   MeSH
• Unfolded Protein Response drug effects   MeSH
• Signal Transduction drug effects   MeSH
• Endoplasmic Reticulum Stress drug effects   genetics   MeSH
• Transcription Factor CHOP genetics   metabolism   MeSH
• Cell Survival drug effects   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Regulated protein synthesis via changes in mRNA structures forms an important part of how prokaryotic cells adapt protein expression in response to changes in the environment. Little is known regarding how this concept has adapted to regulate mRNA translation via signaling pathways in mammalian cells. Here, we show that following phosphorylation by the ataxia telangiectasia mutated (ATM) kinase at serine 403, the C-terminal RING domain of HDMX binds the nascent p53 mRNA to promote a conformation that supports the p53 mRNA-HDM2 interaction and the induction of p53 synthesis. HDMX and its homolog HDM2 bind the same p53 internal ribosome entry sequences (IRES) structure but with different specificity and function. The results show how HDMX and HDM2 act as nonredundant IRES trans-acting factors (ITAFs) to bring a positive synergistic effect on p53 expression during genotoxic stress by first altering the structure of the newly synthesized p53 mRNA followed by stimulation of translation.

• MeSH
• Ataxia Telangiectasia Mutated Proteins metabolism   MeSH
• Phosphorylation MeSH
• Nuclear Proteins chemistry   physiology   MeSH
• Humans MeSH
• RNA, Messenger chemistry   genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Inverted Repeat Sequences MeSH
• DNA Damage MeSH
• RNA Processing, Post-Transcriptional MeSH
• Protein Processing, Post-Translational MeSH
• Protein Biosynthesis MeSH
• Proto-Oncogene Proteins c-mdm2 metabolism   MeSH
• Proto-Oncogene Proteins chemistry   physiology   MeSH
• Gene Expression Regulation MeSH
• RNA Folding MeSH
• Base Sequence MeSH
• Sf9 Cells MeSH
• Spodoptera MeSH
• Substrate Specificity MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The potassium channel protein KCNH2 is encoded by KCNH2 gene, and there are more than 300 mutations of KCNH2. Unfolded protein response (UPR) is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER). The present study aimed to explore the UPR process and the role of activating transcription factor 6 (ATF6) in the abnormal expression of potassium voltage-gated channel subfamily H member 2 (KCNH2)A561V. The wild-type (wt) KCNH2 and A561V mutant KCNH2 was constructed with his-tag. The 293 cells were used and divided into KCNH2wt+KCNH2A561V, KCNH2wt and KCNH2A561V groups. The expression levels of ATF6 and KCNH2 in different groups were detected by Western blotting, reverse transcription-quantitative PCR, immunofluorescence and immuno-coprecipitation assays. The protein types and abundance of immuno-coprecipitation samples were analyzed by mass spectrometry. The proteomic analysis of the mass spectrometry results was carried out by using the reactome database and GO (Gene Ontology) tool. The mRNA expression levels of KCNH2 and ATF6 in the KCNH2wt+KCNH2A561V group were higher compared with the KCNH2A561V group. However, the full-length protein expression of ATF6 was inhibited, indicating that ATF6 was highly activated and a substantial number of ATF6 was sheared in KCNH2wt+KCNH2A561V group compared with control group. Furthermore, A561V-KCNH2 mutation leading to the accumulation of the immature form of KCNH2 (135 kDa bands) in ER, resulting in the reduction of the ratio of 155 kDa/135 kDa. In addition, the abundance of UPR-related proteins in the KCNH2A561V group was higher compared with the KCNH2wt+KCNH2A561V group. The 'cysteine biosynthetic activity' of GO:0019344 process and the 'positive regulation of cytoplasmic translation activity' of GO:2000767 process in the KCNH2A561V group were higher compared with the KCNH2wt+KCNH2A561V group. Hence, co-expression of wild-type and A561V mutant KCNH2 in 293 cells activated the UPR process, which led to the inhibition of protein translation and synthesis, in turn inhibiting the expression of KCNH2. These results provided a theoretical basis for clinical treatment of Long QT syndrome.

• MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Mutation MeSH
• Proteomics * MeSH
• Unfolded Protein Response genetics   MeSH
• Activating Transcription Factor 6 * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH

High pressure methods have become a useful tool for studying protein structure and stability. Using them, various physico-chemical processes including protein unfolding, aggregation, oligomer dissociation or enzyme-activity decrease were studied on many different proteins. Oligomeric protein dissociation is a process that can perfectly utilize the potential of high-pressure techniques, as the high pressure shifts the equilibria to higher concentrations making them better observable by spectroscopic methods. This can be especially useful when the oligomeric form is highly stable at atmospheric pressure. These applications may be, however, hindered by less intensive experimental response as well as interference of the oligomerization equilibria with unfolding or aggregation of the subunits, but also by more complex theoretical description. In this study we develop mathematical models describing different kinds of oligomerization equilibria, both closed (equilibrium of monomer and the highest possible oligomer without any intermediates) and consecutive. Closed homooligomer equilibria are discussed for any oligomerization degree, while the more complex heterooligomer equilibria and the consecutive equilibria in both homo- and heterooligomers are taken into account only for dimers and trimers. In all the cases, fractions of all the relevant forms are evaluated as functions of pressure and concentration. Significant points (inflection points and extremes) of the resulting transition curves, that can be determined experimentally, are evaluated as functions of pressure and/or concentration. These functions can be further used in order to evaluate the thermodynamic parameters of the system, i.e. atmospheric-pressure equilibrium constants and volume changes of the individual steps of the oligomer-dissociation processes.

• MeSH
• Algorithms * MeSH
• Models, Chemical * MeSH
• Kinetics MeSH
• Protein Multimerization * MeSH
• Protein Aggregation, Pathological MeSH
• Protein Subunits chemistry   MeSH
• Proteins chemistry   MeSH
• Protein Unfolding MeSH
• Thermodynamics MeSH
• Pressure MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Castration-resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically-supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol-abuse drug Antabuse) and its copper-chelating metabolite CuET that inhibit protein turnover. METHODS: Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio-resistant stem-cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting. RESULTS: We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR-mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio-resistance of stem-cell like DU145-derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho-eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti-apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR-induced toxicity, yielding synergistic effects of CuET and YM155. CONCLUSIONS: We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.

• MeSH
• PC-3 Cells MeSH
• Molecular Targeted Therapy methods   MeSH
• Disulfiram therapeutic use   MeSH
• PTEN Phosphohydrolase genetics   MeSH
• Stress, Physiological drug effects   genetics   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Prostatic Neoplasms drug therapy   MeSH
• DNA Repair drug effects   MeSH
• Poly(ADP-ribose) Polymerase Inhibitors therapeutic use   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Recombinational DNA Repair drug effects   MeSH
• Radiation Tolerance MeSH
• Vorinostat therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Bone lengthening and fracture repair depend on the anabolic properties of chondrocytes that function in an avascular milieu. The limited supply of oxygen and nutrients calls into question how biosynthesis and redox homeostasis are guaranteed. Here we show that glucose metabolism by the pentose phosphate pathway (PPP) is essential for endochondral ossification. Loss of glucose-6-phosphate dehydrogenase in chondrocytes does not affect cell proliferation because reversal of the non-oxidative PPP produces ribose-5-phosphate. However, the decreased NADPH production reduces glutathione recycling, resulting in decreased protection against the reactive oxygen species (ROS) produced during oxidative protein folding. The disturbed proteostasis activates the unfolded protein response and protein degradation. Moreover, the oxidative stress induces ferroptosis, which, together with altered matrix properties, results in a chondrodysplasia phenotype. Collectively, these data show that in hypoxia, the PPP is crucial to produce reducing power that confines ROS generated by oxidative protein folding and thereby controls proteostasis and prevents ferroptosis.

• MeSH
• Chondrocytes * metabolism   MeSH
• Ferroptosis * physiology   MeSH
• Glucosephosphate Dehydrogenase metabolism   MeSH
• Glucose metabolism   MeSH
• Mice MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress * MeSH
• Pentose Phosphate Pathway * MeSH
• Reactive Oxygen Species * metabolism   MeSH
• Protein Folding * MeSH
• Unfolded Protein Response MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

It is now accepted that reactive oxygen species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS-controlled production is played by the NADPH oxidases (NOXs), a group of seven membrane-bound enzymes (NOX1-5 and DUOX1-2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family member present in cyanobacteria, protists, plants, fungi, and the animal kingdom. We show that the calmodulin-like regulatory EF-domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C-terminal lobe of the EF-domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase (DH) domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the DH domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded-to-folded transition of the EF-domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation.

• MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• NADPH Oxidase 5 chemistry   genetics   metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Cyanobacteria enzymology   MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH