ATP5B Expression Research Articles

The downregulation of NSUN5 may contribute to preeclampsia†.

Preeclampsia (PE) is a complication of pregnancy characterized by the new onset of hypertension after 20weeks of gestation. The incidence of PE is steadily rising, posing a significant threat to the lives of both the pregnant woman and the fetus. Most studies on PE pathogenesis currently focus on the placenta, but maternal decidualization forms the foundation for placental growth and development. Recent studies have shown that impaired decidualization is also a cause of PE. Decidualization is a process where endometrial stromal cells gradually transform into secretory decidual cells during early pregnancy. While NSUN5 encodes a member of a conserved family of proteins, its role in pregnancy remains unknown. In this study, we conducted experiments and observed a significant downregulation of NSUN5 expression in severe PE decidual tissues compared to those of normal pregnant women. When inducing decidualization in vitro, we found an increase in NSUN5 expression. However, when we used siRNA to knockdown NSUN5 expression, the process of decidualization was prevented. Moreover, we observed a decrease in ATP content during both cell decidualization and after knockdown of NSUN5. Finally, through immunoprecipitation combined with mass spectrometry, we discovered that the protein ATP5B interacts with NSUN5. Furthermore, after knocking down ATP5B using siRNA, we observed impaired decidualization. Moreover, transfection with siRNA to suppress NSUN5 resulted in a decrease in ATP5B expression. These significant findings provide strong evidence that NSUN5 plays a crucial role in decidualization and is closely associated with the development of PE through its interaction with ATP5B.

1,4-dihydroxy quininib modulates the secretome of uveal melanoma tumour explants and a marker of oxidative phosphorylation in a metastatic xenograft model.

Uveal melanoma (UM) is an intraocular cancer with propensity for liver metastases. The median overall survival (OS) for metastatic UM (MUM) is 1.07 years, with a reported range of 0.84-1.34. In primary UM, high cysteinyl leukotriene receptor 1 (CysLT1) expression associates with poor outcomes. CysLT1 antagonists, quininib and 1,4-dihydroxy quininib, alter cancer hallmarks of primary and metastatic UM cell lines in vitro. Here, the clinical relevance of CysLT receptors and therapeutic potential of quininib analogs is elaborated in UM using preclinical in vivo orthotopic xenograft models and ex vivo patient samples. Immunohistochemical staining of an independent cohort (n = 64) of primary UM patients confirmed high CysLT1 expression significantly associates with death from metastatic disease (p = 0.02; HR 2.28; 95% CI 1.08-4.78), solidifying the disease relevance of CysLT1 in UM. In primary UM samples (n = 11) cultured as ex vivo explants, 1,4-dihydroxy quininib significantly alters the secretion of IL-13, IL-2, and TNF-α. In an orthotopic, cell line-derived xenograft model of MUM, 1,4-dihydroxy quininib administered intraperitoneally at 25 mg/kg significantly decreases ATP5B expression (p = 0.03), a marker of oxidative phosphorylation. In UM, high ATP5F1B is a poor prognostic indicator, whereas low ATP5F1B, in combination with disomy 3, correlates with an absence of metastatic disease in the TCGA-UM dataset. These preclinical data highlight the diagnostic potential of CysLT1 and ATP5F1B in UM, and the therapeutic potential of 1,4-dihydroxy quininib with ATP5F1B as a companion diagnostic to treat MUM.

Well-of-the-well (WOW) versus polyester mesh (PM): a comparison of single-embryo culture systems in bovines

Background: Mexico is innovating in the livestock industry through in vitro generation of bovine embryos with technologies such as well-of-the-well (WOW) and polyester mesh (PM) single-embryo culture systems. These techniques allow to maintain embryos in separate areas of a shared culture medium. Objective: To compare the quantity and quality of bovine embryos produced in WOW and PM culture systems versus the conventional (CG) culture system. Methods: In total, 345 embryos fertilized in vitro were evaluated for blastocyst yield in the three culture systems. To count blastocyst cell numbers, 69 embryos in each system were differentially stained for trophectoderm (TE), inner cell mass (ICM), and apoptotic cells. A qPCR gene expression analysis was performed for embryos in all three systems. Results: The WOW, PM and CG systems developed similar amount of blastocysts (41, 35 and 36%, respectively; p>0.05). Blastocysts in all three systems showed adequate amounts of ICM and apoptotic cells. Blastocysts in the PM system showed a greater number of TE cells [63.7 versus 58.6% in the CG system (p<0.05)]. Relative mRNA expression of the embryonic genes POUF5F1, GNAS and TP53 did not differ significantly among systems (p>0.05). The ATP5B expression was higher in WOW than in PM (p<0.05), but similar to CG (p>0.05). The TJP3 expression was higher in PM than in WOW and CG (p<0.05). Expression of ID2 and CLDN4 was higher in WOW than in PM and CG (p<0.05). The biplot graphic from Principal Component Analysis (PCA) revealed that CG was located near degenerated embryos, whereas PM was located near arrested embryos, larger ICM and TE, and TJP3 expression. The WOW was located toward blastocysts, morulae, and expression of CLDN4, ID2 and GNAS. Conclusion: Compared with CG, both the PM and WOW systems are good options for culturing single embryos in the bovine model. Moreover, the PCA results suggest that embryos developed in the WOW system have greater capacity for generating blastocysts with increased ability to form TE and ICM layers, which might improve implantation.

Integrated Analyses Reveal the Multi-Omics and Prognostic Characteristics of ATP5B in Breast Cancer.

The beta subunit of F1Fo-ATP synthase (ATP5B) has been demonstrated to play an essential role in tumor progression and metastasis. However, there has been no comprehensive pan-cancer multi-omics analysis of ATP5B, while the clinical relevance of ATP5B and its potential mechanism in regulating breast cancer are still poorly understood. In this study, we demonstrated that ATP5B has a higher frequency of amplification than deletion in most cancer types, and the copy number variation (CNV) of ATP5B was significantly positively correlated with its mRNA expression level. DNA methylation analysis across pan-cancer also revealed a strong correlation between ATP5B expression and epigenetic changes. We identified 6 significant methylation sites involved in the regulation of ATP5B expression. Tissue microarrays (TMA) from 129 breast cancer samples, integrated with multiple additional breast cancer dataset, were used to evaluate the ATP5B expression and its correlation with prognosis. Higher levels of ATP5B expression were consistently associated with a worse OS in all datasets, and Cox regression analysis suggested that ATP5B expression was an independent prognostic factor. Gene enrichment analysis indicated that the gene signatures of DNA damage recognition, the E-cadherin nascent pathway and the PLK1 pathway were enriched in ATP5B-high patients. Moreover, somatic mutation analysis showed that a significant different mutation frequency of CDH1 and ADAMTSL3 could be observed between the ATP5B-high and ATP5B-low groups. In conclusion, this study reveals novel significance regarding the genetic characteristics and clinical value of ATP5B highlighted in predicting the outcome of breast cancer patients.

The function of Cav-1 in MDA-MB-231 breast cancer cell migration and invasion induced by ectopic ATP5B

Ectopic ATP5B, which is located in a unique type of lipid raft caveolar structure, can be upregulated by cholesterol loading. As the structural component of caveolae, Cav-1 is a molecular hub that is involved in transmembrane signaling. In a previous study, the ATP5B-specific binding peptide B04 was shown to inhibit the migration and invasion of prostate cancer cells, and the expression of ATP5B on the plasma membrane of MDA-MB-231 cells was confirmed. The present study investigated the effect of ectopic ATP5B on the migration and invasion of MDA-MB-231 cells and examined the involvement of Cav-1. Cholesterol loading increased the level of ectopic ATP5B and promoted cell migration and invasion. These effects were blocked by B04. Ectopic ATP5B was physically colocalized with Cav-1, as demonstrated by double immunofluorescence staining and coimmunoprecipitation. After Cav-1 knockdown, the migration and invasion abilities of MDA-MB-231 cells were significantly decreased, suggesting that Cav-1 influences the function of ectopic ATP5B. Furthermore, these effects were not reversed after treatment with cholesterol. We concluded that Cav-1 may participate in MDA-MB-231 cell migration and invasion induced by binding to ectopic ATP5B.

ATP5B promotes the metastasis and growth of gastric cancer by activating the FAK/AKT/MMP2 pathway.

Adenosine triphosphate (ATP) in the tumor microenvironment serves a vital role during tumor progression. ATP synthase F1 β subunit (ATP5B) is one of the most important subunits of ATP synthase and increases cellular ATP levels. ATP5B reportedly participates in carcinogenesis in several tumors. However, the regulatory mechanisms of ATP5B remain poorly understood in gastric cancer (GC). Here, we determined that high ATP5B expression in tumor tissues of GC is positively correlated with age, the tumor size, the TNM stage, lymph node metastasis, and patients' poor prognosis. The overexpression of ATP5B in GC cells elevated the cellular ATP content and promoted migration, invasion and proliferation. The levels of MMP2 expression, phosphorylated FAK, and phosphorylated AKT were increased after ATP5B overexpression in GC cells. Additionally, ATP5B overexpression increased the extracellular ATP level through the secretion of intracellular ATP and activated the FAK/AKT/MMP2 signaling pathway. ATP5B-induced downstream pathway activation was induced through the plasma membrane P2X7 receptor. Inhibitors of P2X7, FAK, AKT, and MMP2 suppressed the proliferative, migratory, and invasive capabilities of GC cells. In conclusion, our experiments indicate that ATP5B contributes to tumor progression of GC via FAK/AKT/MMP2 pathway. ATP5B, therefore, may be a biomarker of poor prognosis and a potential therapeutic target for GC.

Role of ATP5B in colorectal cancer

AbstractObjectiveTo explore the role and possible regulatory mechanisms of ATP5B in colorectal cancer.MethodsOnline databases were used to analyze expression of ATP5B and prognosis in patients with l colorectal cancer. The data of TCGA database were analyzed by Gene Set Enrichment Analysis. Co‐expression network analysis of ATP5B was analyzed by coexpedia. Colorectal cancer cell (SW480 and DLD1) were transfected with small interfering RNA and validated by real‐time fluorescence quantitative PCR. Cell proliferation ability was detected by CCK8 experiment, cell migration and invasion ability were detected by scratch experiment and Transwell experiment.ResultsATP5B is highly expressed in colorectal cancer tissue. Up‐regulation of ATP5B is associated with poor prognosis of colorectal cancer patients. ATP5B may regulate the cell junction pathway and the PI3K‐AKT‐mTOR signaling pathway. ATP5B expression is positively correlated with the proliferation, migration, and invasion of colorectal cancer cells.ConclusionsATP5B plays an important role in the tumorigenesis and metastasis of colorectal cancer.

Targeted inhibition of ATP5B gene prevents bone erosion in collagen-induced arthritis by inhibiting osteoclastogenesis

Bone resorption by osteoclasts is an energy consuming activity, which depends on mitochondrial ATP. ATP5B, a mitochondrial ATP synthase beta subunit, is a catalytic core involved in producing ATP. Here, we investigated the contribution of ATP5B in osteoclast differentiation and joint destruction. ATP5B (LV-ATP5B) targeting or non-targeting (LV-NC) siRNA containing lentivirus particles were transduced into bone marrow macrophage derived osteoclasts or locally administered to arthritic mouse joints. Inhibition of ATP5B reduced the expression of osteoclast related genes and proteins, suppressed bone resorption by significantly impairing F-actin formation and decreased the levels of adhesion-associated proteins. In addition, ATP5B deficiency caused osteoclast mitochondrial dysfunction and, impaired the secretion of vacuole protons and MMP9. Importantly, inhibition of ATP5B expression, protected arthritis mice from joint destructions although serum levels of inflammatory mediators (TNF-α, IL-1β) and IgG2α antibodies were unaffected. These results demonstrate an essential function of ATP5B in osteoclast differentiation and bone resorption, and suggest it as a potential therapeutic target for protecting bones in RA.

ATP synthase modulation leads to an increase of spare respiratory capacity in HPV associated cancers

Mucosal and skin cancers are associated with infections by human papillomaviruses (HPV). The manner how viral oncoproteins hijack the host cell metabolism to meet their own energy demands and how this may contribute to tumorigenesis is poorly understood. We now show that the HPV oncoprotein E7 of HPV8, HPV11 and HPV16 directly interact with the beta subunit of the mitochondrial ATP-synthase (ATP5B), which may therefore represent a conserved feature across different HPV genera. By measuring both glycolytic and mitochondrial activity we observed that the association of E7 with ATP5B was accompanied by reduction of glycolytic activity. Interestingly, there was a drastic increase in spare mitochondrial respiratory capacity in HPV8-E7 and an even more profound increase in HPV16-E7 expressing cells. In addition, we could show that ATP5B levels were unchanged in betaHPV positive skin cancers. However, comparing HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas (OPSCC) we noticed that, while ATP5B expression levels did not correlate with patient overall survival in HPV-negative OPSCC, there was a strong correlation within the HPV16-positive OPSCC patient group. These novel findings provide evidence that HPV targets the host cell energy metabolism important for viral life cycle and HPV-mediated tumorigenesis.

Mitotane induces mitochondrial membrane depolarization and apoptosis in thyroid cancer cells.

Mitotane is used for the treatment of adrenocortical cancer and elicits its anticancer effects via inhibition of mitochondrial respiration. Targeting mitochondria-dependent metabolism has emerged as a promising strategy for thyroid cancer (TC) treatment. We hypothesized that mitotane targets mitochondria and induces apoptosis in TC cells. Cell lines representative of the major histological variants of TC were chosen: Follicular (FTC-133), poorly differentiated (BCPAP), anaplastic (SW1736 and C643) and medullary (TT) TC cells, and were treated with mitotane (0-100 μM). Mitochondrial membrane potential, cell viability and apoptosis were examined by JC-1 staining and by western blot analysis using an antibody against caspase-3. The expression of mitochondrial molecules and DNA damage markers and the activation of endoplasmic reticulum (ER) stress were determined by western blotting. The expression of mitochondrial ATP synthase subunit β (ATP5B) was examined by immunostaining in 100 human TC tissue samples. Treatment with mitotane (50 μM for 24 h) decreased the viability of FTC-133, BCPAP, SW1736, C643 and TT cells by 12, 59, 54, 31 and 66%, respectively. Morphological evidence of ER stress and overexpression of ER markers was observed in TC cells following exposure to mitotane. The treatment led to increased expression of histone γH2AX, indicating DNA damage, and to caspase-3 cleavage. Consistent with the results of the cell viability assays, the overexpression of pro-apoptotic genes following treatment with mitotane was more prominent in TC cells harboring mutations in the serine/threonine-protein kinase B-raf gene and proto-oncogene tyrosine-protein kinase receptor Ret. Treatment with mitotane was associated with loss of mitochondrial membrane potential and decreased expression of ATP5B, particularly in the medullary TC (MTC)-derived TT cells. Immunohistochemical analysis of mitochondrial ATP5B in human TC specimens demonstrated its overexpression in cancer compared with normal thyroid tissue. The level of ATP5B expression was higher in MTC compared with the follicular, papillary or anaplastic types of TC. Mitotane elicited pleiotropic effects on TC cells, including induction of ER stress, inhibition of mitochondrial membrane potential and induction of apoptosis. The results of the present study suggest that mitotane could be considered as a novel agent for the treatment of aggressive types of TC.

A novel RNA aptamer identifies plasma membrane ATP synthase beta subunit as an early marker and therapeutic target in aggressive cancer

PurposePrimary breast and prostate cancers can be cured, but metastatic disease cannot. Identifying cell factors that predict metastatic potential could guide both prognosis and treatment.MethodsWe used Cell-SELEX to screen an RNA aptamer library for differential binding to prostate cancer cell lines with high vs. low metastatic potential. Mass spectroscopy, immunoblot, and immunohistochemistry were used to identify and validate aptamer targets. Aptamer properties were tested in vitro, in xenograft models, and in clinical biopsies. Gene expression datasets were queried for target associations in cancer.ResultsWe identified a novel aptamer (Apt63) that binds to the beta subunit of F1Fo ATP synthase (ATP5B), present on the plasma membrane of certain normal and cancer cells. Apt63 bound to plasma membranes of multiple aggressive breast and prostate cell lines, but not to normal breast and prostate epithelial cells, and weakly or not at all to non-metastasizing cancer cells; binding led to rapid cell death. A single intravenous injection of Apt63 induced rapid, tumor cell-selective binding and cytotoxicity in MDA-MB-231 xenograft tumors, associated with endonuclease G nuclear translocation and DNA fragmentation. Apt63 was not toxic to non-transformed epithelial cells in vitro or adjacent normal tissue in vivo. In breast cancer tissue arrays, plasma membrane staining with Apt63 correlated with tumor stage (p < 0.0001, n = 416) and was independent of other cancer markers. Across multiple datasets, ATP5B expression was significantly increased relative to normal tissue, and negatively correlated with metastasis-free (p = 0.0063, 0.00039, respectively) and overall (p = 0.050, 0.0198) survival.ConclusionEcto-ATP5B binding by Apt63 may disrupt an essential survival mechanism in a subset of tumors with high metastatic potential, and defines a novel category of cancers with potential vulnerability to ATP5B-targeted therapy. Apt63 is a unique tool for elucidating the function of surface ATP synthase, and potentially for predicting and treating metastatic breast and prostate cancer.

Isobaric tags for relative and absolute quantitation‑based proteomics reveals potential novel biomarkers for the early diagnosis of acute myocardial infarction within 3h.

Acute myocardial infarction (AMI) is one of the most common and life-threatening cardiovascular diseases. However, the ability to diagnose AMI within 3 h is currently lacking. The present study aimed to identify the differentially expressed proteins of AMI within 3 h and to investigate novel biomarkers using isobaric tags for relative and absolute quantitation (ITRAQ) technology. A total of 30 beagle dogs were used for establishing the MI models successfully by injecting thrombin powder and a polyethylene microsphere suspension. Serum samples were collected prior to (0 h) and following MI (1, 2 and 3 h). ITRAQ-coupled liquid chromatography-mass spectrometry (LC-MS) technology was used to identify the differentially expressed proteins. The bioinformatics analysis selected several key proteins in the initiation of MI. Further analysis was performed using STRING software. Finally, western blot analysis was used to evaluate the results obtained from ITRAQ. In total, 28 proteins were upregulated and 23 were downregulated in the 1 h/0 h group, 28 proteins were upregulated and 26 were downregulated in the 2 h/0 h group, and 24 proteins were upregulated and 19 were downregulated in the 3 h/0 h group. The Gene Ontology (GO) annotation and functional enrichment analysis identified 19 key proteins. Protein-protein interactions (PPIs) were investigated using the STRING database. GO enrichment analysis revealed that a number of key proteins, including ATP synthase F1 subunit β (ATP5B), cytochrome c oxidase subunit 2 and cytochrome c, were components of the electron transport chain and were involved in energy metabolism. The western blot analysis demonstrated that the expression of ATP5B decreased significantly at all three time points (P<0.01), which was consistent with the ITRAQ results, whereas the expression of fibrinogen γ chain increased at 2 and 3 h (P<0.01) and the expression of integrator complex subunit 4 increased at all three time points (P<0.01), which differed from the ITRAQ results. According to the proteomics of the beagle dog MI model, ATP5B may serve as the potential biomarkers of AMI. Mitochondrial dysfunction and disruption of the electron transport chain may be critical indicators of early MI within 3 h. These finding may provide a novel direction for the diagnosis of AMI.

FUS interacts with ATP synthase beta subunit and induces mitochondrial unfolded protein response in cellular and animal models

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPRmt). Importantly, down-regulating expression of ATP5B or UPRmt genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration.

Possible Involvement of F1F0-ATP synthase and Intracellular ATP in Keratinocyte Differentiation in normal skin and skin lesions

The F1F0-ATP synthase, an enzyme complex, is mainly located on the mitochondrial inner membrane or sometimes cytomembrane to generate or hydrolyze ATP, play a role in cell proliferation. This study focused on the role of F1F0-ATP synthase in keratinocyte differentiation, and its relationship with intracellular and extracellular ATP (InATP and ExATP). The F1F0-ATP synthase β subunit (ATP5B) expression in various skin tissues and confluence-dependent HaCaT differentiation models was detected. ATP5B expression increased with keratinocyte and HaCaT cell differentiation in normal skin, some epidermis hyper-proliferative diseases, squamous cell carcinoma, and the HaCaT cell differentiation model. The impact of InATP and ExATP content on HaCaT differentiation was reflected by the expression of the differentiation marker involucrin. Inhibition of F1F0-ATP synthase blocked HaCaT cell differentiation, which was associated with a decrease of InATP content, but not with changes of ExATP. Our results revealed that F1F0-ATP synthase expression is associated with the process of keratinocyte differentiation which may possibly be related to InATP synthesis.

ATP synthase subunit-β down-regulation aggravates diabetic nephropathy.

In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition.

Investigation of trichloroethylene-induced effects on subcellular proteomes in L-02 hepatic cells

To put the insight into the trichloroethylene (TCE)-induced effect on the differential expression of subcellular proteins in human normal liver cell line (L-02). The membrane proteins and nuclear proteins of TCE-treated (8.0 mmol/L) group and controls were extracted by subcellular proteome extraction kit, respectively. The TCE-induced differentially expressions were analyzed by a two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight spectrometry (MALDI-TOF-MS). Bioinformatics analysis was used to reveal the biological processes and predict transmembrane domains of differential expressed proteins. The expression of ATP synthase subunit beta (ATP5B), heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) and far up steam element-binding protein 1 (FUBP1) were measured under TCE treatment by Western blot. After TCE treatment for 24 h in L-02 cells, 14 membrane proteins and 18 nuclear proteins were identified as differential expression. After treated with TCE in concentrations of 0, 2.0, 4.0 and 8.0 mmol/L for 24 h, the relative levels of ATP5B expression were 1.00±0.03, 1.21±0.14, 1.25±0.12 and 1.48±0.17 (F = 8.51, P = 0.007), the relative levels of hnRNP H2 expression were 1.00±0.09, 1.22±0.15, 1.43±0.21, 1.53±0.17 (F = 6.57, P = 0.015), respectively; the relative levels of FUBP1 expression were 1.00±0.11, 0.91±0.07, 0.73±0.04 and 0.67±0.03 (F = 15.81, P = 0.001), respectively, which were consistent with the results in proteomics. The bioinformatics analysis showed that the most dominant biological process were involved in RNA processing (10 proteins, P = 2.46×10(-6)), especially in RNA splicing (9 proteins, P = 1.77×10(-7)). The exposure of TCE could alter the expression of membrane proteins and nuclear proteins in L-02 cells. These abnormal expressed proteins involved in RNA splicing would provide novel clues for further understanding of TCE-induced hepatotoxicity.

ATP5b and β2-microglobulin are predictive markers for the prognosis of patients with gallbladder cancer.

The differences in clinical, pathological, and biological characteristics between adenocarcinoma (AC) and squamous cell/adenosquamous carcinoma (SC/ASC) of the gallbladder have not been well documented. This study investigates the clinical and pathological associations of ATP5B and β2M with benign and malignant lesions of the gallbladder. In this study, ATP5B and β2M expression in 46 SC/ASCs and 80 ACs were examined using immunohistochemistry. The rate of ATP5B positive expression was significantly lower, while the rate of β2M expression was significantly higher, in AC and SC/ASC than in gallbladder adenomas, gallbladder polyps, or gallbladder epithelium with stone (P < 0.01). More SC/ASCs had larger tumor mass and good differentiation compared to ACs. Positive β2M and negative ATP5B expression were significantly associated with large tumor size, high TNM stage, lymph node metastasis, and invasion of SC/ASCs and ACs. Univariate Kaplan-Meier analysis showed that positive β2M (P < 0.05 or P < 0.001) expression and negative ATP5B (P < 0.001) expression were significantly associated with decreased overall survival in both SC/ASC and AC patients. Multivariate Cox regression analysis showed that negative ATP5B expression is an independent-prognostic factor for poor prognosis in both SC/ASC (P < 0.01) and AC (P < 0.001) patients. Positive β2M expression is an independent-prognostic factor for poor prognosis in AC (P < 0.05) patients. Our study suggested that positive β2M expression or loss of ATP5B expression in tumor tissues is closely related to the metastasis, invasion, and poor-prognosis of gallbladder cancer.

Altered Mitochondrial Function and Energy Metabolism Is Associated with a Radioresistant Phenotype in Oesophageal Adenocarcinoma

Neoadjuvant chemoradiation therapy (CRT) is increasingly the standard of care for locally advanced oesophageal cancer. A complete pathological response to CRT is associated with a favourable outcome. Radiation therapy is important for local tumour control, however, radioresistance remains a substantial clinical problem. We hypothesise that alterations in mitochondrial function and energy metabolism are involved in the radioresistance of oesophageal adenocarcinoma (OAC). To investigate this, we used an established isogenic cell line model of radioresistant OAC. Radioresistant cells (OE33 R) demonstrated significantly increased levels of random mitochondrial mutations, which were coupled with alterations in mitochondrial function, size, morphology and gene expression, supporting a role for mitochondrial dysfunction in the radioresistance of this model. OE33 R cells also demonstrated altered bioenergetics, demonstrating significantly increased intracellular ATP levels, which was attributed to enhanced mitochondrial respiration. Radioresistant cells also demonstrated metabolic plasticity, efficiently switching between the glycolysis and oxidative phosphorylation energy metabolism pathways, which were accompanied by enhanced clonogenic survival. This data was supported in vivo, in pre-treatment OAC tumour tissue. Tumour ATP5B expression, a marker of oxidative phosphorylation, was significantly increased in patients who subsequently had a poor pathological response to neoadjuvant CRT. This suggests for the first time, a role for specific mitochondrial alterations and metabolic remodelling in the radioresistance of OAC.

METTL23, a transcriptional partner of GABPA, is essential for human cognition.

Whereas many genes associated with intellectual disability (ID) encode synaptic proteins, transcriptional defects leading to ID are less well understood. We studied a large, consanguineous pedigree of Arab origin with seven members affected with ID and mild dysmorphic features. Homozygosity mapping and linkage analysis identified a candidate region on chromosome 17 with a maximum multipoint logarithm of odds score of 6.01. Targeted high-throughput sequencing of the exons in the candidate region identified a homozygous 4-bp deletion (c.169_172delCACT) in the METTL23 (methyltransferase like 23) gene, which is predicted to result in a frameshift and premature truncation (p.His57Valfs*11). Overexpressed METTL23 protein localized to both nucleus and cytoplasm, and physically interacted with GABPA (GA-binding protein transcription factor, alpha subunit). GABP, of which GABPA is a component, is known to regulate the expression of genes such as THPO (thrombopoietin) and ATP5B (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide) and is implicated in a wide variety of important cellular functions. Overexpression of METTL23 resulted in increased transcriptional activity at the THPO promoter, whereas knockdown of METTL23 with siRNA resulted in decreased expression of ATP5B, thus revealing the importance of METTL23 as a regulator of GABPA function. The METTL23 mutation highlights a new transcriptional pathway underlying human intellectual function.

Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas

Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.