Mitophagy is involved in the mitochondrial dysfunction of vitrified porcine oocytes.

Mitochondrial dysfunction is considered a crucial factor aggravating oocyte viability after vitrification-warming. To clarify the role of mitophagy in mitochondrial extinction of vitrified porcine oocytes, mitochondrial function, ultrastructural characteristics, mitochondria-lysosomes colocalization, and mitophagic proteins were detected with or without chloroquine (CQ) treatment. The results showed that vitrification caused mitochondrial dysfunction, including increasing reactive oxygen species production, decreasing mitochondrial membrane potential, and mitochondrial DNA copy number. Damaged mitochondrial cristae and mitophagosomes were observed in vitrified oocytes. A highly fused fluorescence distribution of mitochondria and lysosomes was also observed. In the detection of mitophagic flux, mitophagy was demonstrated as increasing fluorescence aggregation of microtubule-associated protein light chain 3B (LC3B), enhanced colocalization between LC3B, and voltage-dependent anion channels 1 (VDAC1), and upregulated LC3B-II/I protein expression ratio. CQ inhibited the degradation of mitophagosomes in vitrified oocytes, manifested as decreased mitochondria-lysosomes colocalization, increased fluorescence fraction of VDAC1 overlapping LC3B, increased LC3B-II/I protein expression ratio, and p62 accumulation. The inhibition of mitophagosomes degradation by CQ aggravated mitochondrial dysfunction, including increased oxidative damage, reduced mitochondrial function, and further led to loss of oocyte viability and developmental potentiality. In conclusion, mitophagy is involved in the regulation of mitochondrial function during porcine oocyte vitrification.

Detergent Titration as an Efficient Method for NMR Resonance Assignments of Membrane Proteins in Lipid-Bilayer Nanodiscs.

Lipid bilayer nanodiscs are an attractive tool to study membrane proteins in a detergent-free lipid-bilayer environment. In the case of NMR studies, a sequence-specific resonance assignment is required in order to gain structural and functional insights with atomic resolution. Although NMR backbone assignments of membrane proteins in detergents are available, they are largely absent for membrane proteins in nanodiscs due to unfavorable relaxation properties of the slowly tumbling membrane protein-nanodisc complex. The necessary residue-specific reassignment of resonances in nanodiscs is therefore extremely time and sample consuming and represents the fundamental bottleneck in the application of nanodiscs for NMR studies. Here we present an elegant and fast solution to the problem. We show that a resonance assignment in detergent micelles can be transferred to a spectrum recorded in nanodiscs via detergent titration. The procedure requires that lipid-detergent exchange kinetics are in the fast exchange regime in order to follow linear and nonlinear peak shift trajectories with increasing detergent concentration. We demonstrate the feasibility of the approach on the 148-residue membrane protein OmpX. The titration method is then applied to VDAC, a 19-stranded ß-barrel with 283 residues, for which 67% of the detergent assignment could be transferred to the nanodisc spectrum. We furthermore show that this method also works for the largest currently assigned membrane protein, BamA with 398 residues. The method is applicable for backbone amide and side chain methyl groups and represents a time and cost-effective assignment method, for example, to investigate membrane protein allostery and drug binding in a more natural and detergent-free lipid bilayer.

VDAC1, mitochondrial dysfunction, and Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Although an accumulation of brain amyloid-ß (Aß) peptide and hyperphosphorylated tau protein have been implicated in the pathogenesis of AD, the etiology of the disease remains unclear. Mitochondrial dysfunction has been identified as an early event in AD pathogenesis and is reflected by reduced metabolism, disruption of Ca2+ homeostasis, and increased levels of reactive oxygen species, lipid peroxidation, and apoptosis. The focus of this review is the involvement of mitochondrial dysfunction in AD, and specifically, the role of the voltage-dependent anion channel 1 (VDAC1), which has been linked to AD pathogenesis. VDAC1 is a multi-functional protein, expressed in the mitochondria and other cell compartments, including the plasma membrane. The protein regulates the main metabolic and energetic functions of the cell, including Ca2+ homeostasis, oxidative stress, and mitochondria-mediated apoptosis. VDAC1 represents a hub protein that interacts with over 150 other proteins including phosphorylated tau, Aß, and γ-secretase, and participates in their toxicity. The high levels of VDAC1 demonstrated post-mortem in the brains of AD patients and in amyloid precursor protein (APP) transgenic mice prompted the hypothesis that the protein may be associated with neuronal cell destruction since over-expression of VDAC1 triggers cell death. Thus, targeting mitochondrial dysfunction via VDAC1, to prevent this pro-apoptotic activity, could represent a novel strategy for inhibiting cell death. In addition, the review also discusses possible VDAC1 involvement in the link between AD and diabetes and the inverse association between cancer and AD.

Local mitochondrial-endolysosomal microfusion cleaves voltage-dependent anion channel 1 to promote survival in hypoxia.

The oxygen-limiting (hypoxic) microenvironment of tumors induces metabolic reprogramming and cell survival, but the underlying mechanisms involving mitochondria remain poorly understood. We previously demonstrated that hypoxia-inducible factor 1 mediates the hyperfusion of mitochondria by inducing Bcl-2/adenovirus E1B 19-kDa interacting protein 3 and posttranslational truncation of the mitochondrial ATP transporter outer membrane voltage-dependent anion channel 1 in hypoxic cells. In addition, we showed that truncation is associated with increased resistance to drug-induced apoptosis and is indicative of increased patient chemoresistance. We now show that silencing of the tumor suppressor TP53 decreases truncation and increases drug-induced apoptosis. We also show that TP53 regulates truncation through induction of the mitochondrial protein Mieap. While we found that truncation was independent of mitophagy, we observed local microfusion between mitochondria and endolysosomes in hypoxic cells in culture and in patients' tumor tissues. Since we found that the endolysosomal asparagine endopeptidase was responsible for truncation, we propose that it is a readout of mitochondrial-endolysosomal microfusion in hypoxia. These novel findings provide the framework for a better understanding of hypoxic cell metabolism and cell survival through mitochondrial-endolysosomal microfusion regulated by hypoxia-inducible factor 1 and TP53.

Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Impact of exposure to low concentrations of nitric oxide on protein profile in murine and human pancreatic islet cells.

Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic ß cells, but the protein profile of this action remains to be determined. We have applied a 2 dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediated oxidative stress response pathways and eNOS signaling. The results indicate that exposure to exogenous NO counteracts the impact of serum deprivation on pancreatic ß cell proteome. Species differences in the proteins involved are apparent.

MAVS regulates apoptotic cell death by decreasing K48-linked ubiquitination of voltage-dependent anion channel 1.

The mitochondrial antiviral signaling protein MAVS (IPS-1, VISA, or Cardif) plays an important role in the host defense against viral infection by inducing type I interferon. Recent reports have shown that MAVS is also critical for virus-induced apoptosis. However, the mechanism of MAVS-mediated apoptosis induction remains unclear. Here, we show that MAVS binds to voltage-dependent anion channel 1 (VDAC1) and induces apoptosis by caspase-3 activation, which is independent of its role in innate immunity. MAVS modulates VDAC1 protein stability by decreasing its degradative K48-linked ubiquitination. In addition, MAVS knockout mouse embryonic fibroblasts (MEFs) display reduced VDAC1 expression with a consequent reduction of the vesicular stomatitis virus (VSV)-induced apoptosis response. Notably, the upregulation of VDAC1 triggered by VSV infection is completely abolished in MAVS knockout MEFs. We thus identify VDAC1 as a target of MAVS and describe a novel mechanism of MAVS control of virus-induced apoptotic cell death.

Identification of prognostic protein biomarkers in childhood acute lymphoblastic leukemia (ALL).

Early response to 7 days of prednisolone (PRED) treatment is one of the important prognostic factors in predicting eventual outcome in childhood acute lymphoblastic leukemia (ALL). Using proteomic tools and clinically important leukemia cell lines (REH, 697, Sup-B15, RS4; 11), we have identified potential prognostic protein biomarkers as well as discovered promising regulators of PRED-induced apoptosis. After treatment with PRED, the four cell lines can be separated into resistant (REH) and sensitive (697, Sup-B15, RS4;11). Two dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS identified 77 and 17 significantly differentially expressed protein spots (p<0.05) in PRED-sensitive and PRED-resistant cell lines respectively. Several of these were validated by Western blot including proliferating cell nuclear antigen (PCNA), cofilin 1, voltage-dependent anion-channel protein 1 (VDAC1) and proteasome activator subunit 2 (PA28ß). PCNA is a promising protein because of its important roles both in cell cycle regulation and survival control. We subsequently validated PCNA in 43 paired bone marrow samples from children with newly diagnosed ALL (Day 0) and 7 days after PRED treatment (Day 8). ROC curve analysis confirmed that PCNA was highly predictive of PRED response in patients (AUC=0.81, p=0.007) and most interestingly, independent of the molecular subtype, providing a promising universal prognostic marker.

Gene expression meta-analysis identifies VDAC1 as a predictor of poor outcome in early stage non-small cell lung cancer.

BACKGROUND: The bioenergetic status of non-small cell lung cancer correlates with tumour aggressiveness. The voltage dependent anion channel type 1 (VDAC1) is a component of the mitochondrial permeability transition pore, regulates mitochondrial ATP/ADP exchange suggesting that its over-expression could be associated with energy dependent processes including increased proliferation and invasiveness. To test this hypothesis, we conducted an in vivo gene-expression meta-analysis of surgically resected non-small cell lung cancer (NSCLC) using 602 individual expression profiles, to examine the impact of VDAC1 on survival. METHODOLOGY/PRINCIPAL FINDINGS: High VDAC1 expression was associated with shorter overall survival with hazard ratio (HR) = 0.6639 (95% confidence interval (CI) 0.4528 to 0.9721), p = 0.035352 corresponding to 52 versus 101 months. VDAC1 predicted shorter time to recurrence and was shown to be an independent prognostic factor compared with histology, gender, age, nodal stage and tumour stage in a Cox multivariate analysis. Supervised analysis of all the datasets identified a 6-gene signature comprising HNRNPC, HSPA4, HSPA9, UBE2D2, CSNK1A1 and G3BP1 with overlapping functions involving regulation of protein turnover, RAS-RAF-MEK pathway and transcription. VDAC1 predicted survival in breast cancer and myeloma and an unsupervised analysis revealed enrichment of the VDAC1 signature in specific subsets. CONCLUSIONS: In summary, gene expression analysis identifies VDAC1 gene expression as a predictor of poor outcome in NSCLC and other cancers and is associated with dysregulation of a conserved set of biological pathways, which may be causally associated with aggressive tumour behaviour.

A study of association between expression of hOGG1, VDAC1, HK-2 and cervical carcinoma.

BACKGROUND: Human 8-oguanine glycosylase 1(hOGG1), voltage-dependent anion channel 1(VDAC1), hexokinase 2(HK-2), represented the process of oxidative DNA damage, cell apoptosis and glycolysis, respectively. This study aims to explore the association between expression of hOGG1, VDAC1, HK-2 and cervical carcinoma. METHODS: A case-control study was conducted. 65 cervical biopsy samples consist of 20 control and 45 cases. The expression of hOGG1, VDAC1 and HK-2 were examined with immunohistochemistry(IHC), immunolabeling was evaluated with stereological cell counts. RESULTS: The data showed that the positive proportion of hOGG1 and HK-2 in the case group was higher than that of the control group (P < 0.05). Further, there was an increasing trend for the positive proportion and expression degree of hOGG1 and HK-2 from Control, Mild cervical carcinoma (MCC), Intermediate cervical carcinoma(ICC) to Severe cervical carcinoma(SCC) in order (P < 0.05). To VDAC1, the significant result was not obtained. CONCLUSIONS: The results suggested that there was a close association between expression of hOGG1, HK-2 and cervical cancer. hOGG1 and HK-2 might play a key role at the early stage of cervical cancer, and the findings of hOGG1 and HK-2 should be considered as a significant biomarker at the early stage of cervical cancer.

Coupled decomposition of four-dimensional NOESY spectra.

Four-dimensional (4D) NOESY spectra provide unambiguous distance information at a resolution that cannot be achieved in fewer dimensions and thus increase the quality of biomolecular structure determination substantially. Since the degree of chemical shift degeneracy increases with protein size, the use of 4D NOESY spectra is particularly important for large proteins. The potential high resolution in 4D spectra cannot be achieved in a reasonable time with conventional acquisition routines that sample the Nyquist grid uniformly. It can, however, be obtained with nonuniform sampling of the data grid, but optimal processing of such data has not yet been established. Here we describe a processing method for a pair of sparsely sampled 4D NOESY spectra, a methyl-methyl and an amide-methyl NOESY, recorded on a perdeuterated protein with protonated isoleucine, leucine, and valine methyl groups. The coupled multidimensional decomposition (Co-MDD) of these two spectra together with a 2D template spectrum results in a substantial increase in sensitivity, evidenced by 50-100% additional cross peaks, when compared to alternative processing schemes. At the same time, Co-MDD allows the use of low sparse levels of 10-15% of the full data grid for NOESY spectra. For the 283-residue integral human membrane protein VDAC-1, which has a rotational correlation time of about 70 ns in detergent micelles, the two 4D Co-MDD NOESYs yielded a total of 366 NOEs, resulting in 139 unambiguous upper limit distance constraints for the structure calculation.

Expression and localization of voltage-dependent anion channels (VDAC) in human spermatozoa.

Voltage-dependent anion channels (VDAC), also known as mitochondrial porins, are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures. VDAC allow the passage of the metabolites across the mitochondrial outer membrane, and are involved in metabolite transport and signal transduction. Several recent studies have indicated the important roles of VDAC in maintaining normal structure and motility of mammalian spermatozoa. To study the expression and localization of VDAC in human spermatozoa, different experimental approaches were applied: (1) specific primers were designed and VDAC gene sequences were cloned by PCR amplification from human testis cDNA library; (2) recombinant VDAC proteins were produced in the expression vector Escherichia coli BL21 (DE3); (3) human sperm VDAC proteins were extracted, separated and analyzed by Western blotting; (4) the localization of VDAC in human spermatozoa were detected using immunofluorescence. The three gene sequences and recombinant VDAC proteins were obtained, respectively. VDAC proteins were detected to be located in human spermatozoa, especially in sperm flagella. Our study elucidated for the first time that VDAC were synthesized and secreted at the testis level and eventually became an integral part of sperm proteins.

2-DE and MS analysis of interactions between Lactobacillus fermentum I5007 and intestinal epithelial cells.

Lactobacillus is a probiotic commonly used for supplementation to human and animal diets. In this study, we used 2-DE and MS to analyze changes in the proteomes of Lactobacillus and intestinal epithelial cells in two model systems. The in vivo and in vitro models were involved the inoculation of Lactobacillus fermentum I5007 into the rabbit jejunum for 4 h and the culture of the bacterium with Caco-2 cells for 1 h, respectively. Our results indicate that, after exposure to the intestinal environment, the bacterium exhibited decreases in key enzymes involved in energy metabolism (e.g., lactate dehydrogenase, dihydrolipoamide dehydrogenase, and nicotinate phosphoribosyltransferase) and amino acid metabolism (e.g., arginyl-tRNA synthetase and aspartate-semialdehyde dehydrogenase), but increases in glycoside hydrolase (an enzyme for mucin degradation) and fructose-6-phosphate phosphoketolase (an enzyme of the pentose phosphate pathway). In response to an interaction with L. fermentum I5007, Caco-2 cells showed changes in proteins that were beneficial for gut integrity, including voltage-dependent anion channel 1, glutathione transferase, and heat shock protein gp96. On the basis of their functions, we suggest that these proteins serve as useful biomarkers for metabolic changes in Lactobacillus and intestinal epithelial cells in response to their interactions.

Proteomic identification of new biomarkers and application in thyroid cytology.

OBJECTIVE: To validate proteins identified by proteomics as potentially usable markers in thyroid pathology. STUDY DESIGN: Frozen sections of thyroid tumors were manually micro-dissected and proteins extracted. Two-dimensional (2D) gel electrophoresis and subsequent liquid chromatography/mass spectroscopy were performed, and differentially expressed proteins were identified. Validation of candidates for tumor markers (galectin-1, galectin-3, S100C and voltage-dependent anion channel 1 [VDAC1]) was done by immunohistochemistry in 21 cell blocks from fine needle aspiration biopsies (FNAB) and corresponding histology specimens (13 cases). RESULTS: Galectin-3 was negative in benign lesions and positive in FNAB from papillary carcinoma (5 of 5), follicular variant of papillary carcinoma (1 of 4) and follicular carcinoma (1 of 2). S100C was positive in some benign lesions: hyperplasia (2 of 4), goiter (1 of 3) and follicular adenoma (1 of 3), with predominantly nuclear pattern of staining. S100C was positive in malignant lesions, showing cytoplasmic location. Galectin-1 was negative in benign lesions and positive in follicular carcinoma (1 of 2), papillary carcinoma (2 of 5) and follicular variant of papillary carcinoma (1 of 4). VDAC1 was detected in benign and malignant lesions, showing a strong positivity in follicular carcinomas. CONCLUSION: Immunohistochemical validation of potential markers is a crucial step before clinical application in diagnosis. Galectin-3, galectin-1 and S100C can be used to help in discriminating benign and malignant thyroid lesions.

Soy extract has different effects compared with the isolated isoflavones on the proteome of homocysteine-stressed endothelial cells.

Epidemiological studies suggest that soy consumption may provide a protection in the development and progression of atherosclerosis. It is under debate, however, whether the soy isoflavones or other compounds are the "active principle". As apoptosis is a driving force in the process of atherosclerosis, we tested whether a soy extract or a combination of the two predominant isoflavones genistein and daidzein, in concentrations as found in the extract, exert similar or different effects on apoptosis in EA.hy 926 endothelial cells after exposure to the endothelial stressor homocysteine. Plasma membrane disintegration and nuclear fragmentation served as relevant apoptosis markers. To assess whether the extract and the genistein/daidzein mixture differently affect cellular target proteins changed in amount by homocysteine treatment, proteome analysis was performed by two-dimensional gel-electrophoresis and peptide mass fingerprinting of regulated protein spots. Homocysteine induced apoptosis in the cells, and both extract and genistein/daidzein inhibited apoptosis to a comparable extent. Whereas the extract prevented for 10 proteins the changes in expression levels as caused by homocysteine, the genistein/daidzein mixture reversed the homocysteine effects on the proteome for 13 proteins. The cytoskeletal protein matrin 3 and a U5 snRNP-specific 40-kDa protein were the only protein entities where both extract and genistein/daidzein reversed the homocysteine-induced changes in a common way. In conclusion, our studies provide evidence that an isoflavone containing soy extract and isolated isoflavones, despite similar effects on inhibition of homocysteine-induced apoptosis in endothelial cells, affect a quite different spectrum of cellular target proteins.