SLC25A46 promotes mitochondrial fission and mediates resistance to lipotoxic stress in INS-1E insulin-secreting cells.

Glucose sensing in pancreatic ß-cells depends on oxidative phosphorylation and mitochondria-derived signals that promote insulin secretion. Using mass spectrometry-based phosphoproteomics to search for downstream effectors of glucose-dependent signal transduction in INS-1E insulinoma cells, we identified the outer mitochondrial membrane protein SLC25A46. Under resting glucose concentrations, SLC25A46 was phosphorylated on a pair of threonine residues (T44/T45) and was dephosphorylated in response to glucose-induced Ca2+ signals. Overexpression of SLC25A46 in INS-1E cells caused complete mitochondrial fragmentation, resulting in a mild mitochondrial defect associated with lowered glucose-induced insulin secretion. In contrast, inactivation of the Slc25a46 gene resulted in dramatic mitochondrial hyperfusion, without affecting respiratory activity or insulin secretion. Consequently, SLC25A46 is not essential for metabolism-secretion coupling under normal nutrient conditions. Importantly, insulin-secreting cells lacking SLC25A46 had an exacerbated sensitivity to lipotoxic conditions, undergoing massive apoptosis when exposed to palmitate. Therefore, in addition to its role in mitochondrial dynamics, SLC25A46 plays a role in preventing mitochondria-induced apoptosis in INS-E cells exposed to nutrient stress. By protecting mitochondria, SLC25A46 might help to maintain ß-cell mass essential for blood glucose control.

Assessing normalization methods in mass spectrometry-based proteome profiling of clinical samples.

BACKGROUND: Large-scale proteomic studies have to deal with unwanted variability, especially when samples originate from different centers and multiple analytical batches are needed. Such variability is typically added throughout all the steps of a clinical research study, from human biological sample collection and storage, sample preparation, spectral data acquisition, to peptide and protein quantification. In order to remove such diverse and unwanted variability, normalization of the protein data is performed. There have been already several published reviews comparing normalization methods in the -omics field, but reports focusing on proteomic data generated with mass spectrometry (MS) are much fewer. Additionally, most of these reports have only dealt with small datasets. RESULTS: As a case study, here we focused on the normalization of a large MS-based proteomic dataset obtained from an overweight and obese pan-European cohort, where different normalization methods were evaluated, namely: center standardize, quantile protein, quantile sample, global standardization, ComBat, median centering, mean centering, single standard and removal of unwanted variation (RUV); some of these are generic normalization methods while others have been specifically created to deal with genomic or metabolomic data. We checked how relationships between proteins and clinical variables (e.g., gender, levels of triglycerides or cholesterol) were improved after normalizing the data with the different methods. CONCLUSIONS: Some normalization methods were better adapted for this particular large-scale shotgun proteomic dataset of human plasma samples labeled with isobaric tags and analyzed with liquid chromatography-tandem MS. In particular, quantile sample normalization, RUV, mean and median centering showed very good performances, while quantile protein normalization provided worse results than those obtained with unnormalized data.

Proteomes of Paired Human Cerebrospinal Fluid and Plasma: Relation to Blood-Brain Barrier Permeability in Older Adults.

The systems-level relationship between the proteomes of cerebrospinal fluid (CSF) and plasma has not been comprehensively described so far. Recently developed shotgun proteomic workflows allow for deeper characterization of the proteomes from body fluids in much larger sample size. We deployed state-of-the-art mass spectrometry-based proteomics in paired CSF and plasma samples volunteered by 120 elders with and without cognitive impairment to comprehensively characterize and examine compartmental proteome differences and relationships between both body fluids. We further assessed the influence of blood-brain barrier (BBB) integrity and tested the hypothesis that BBB breakdown can be identified from CSF and plasma proteome alterations in nondemented elders. We quantified 790 proteins in CSF and 422 proteins in plasma, and 255 of the proteins were identified in both compartments. Pearson's statistics determined 28 proteins with associated levels between CSF and plasma. BBB integrity as defined with the CSF/serum albumin index influenced 76 CSF/plasma protein ratios. In least absolute shrinkage and selection operator models, CSF and plasma proteins improved identification of BBB impairment. In conclusion, we provide here a first comprehensive draft map of interacting human CSF and plasma proteomes, in view of their complex and dynamic compositions, and influence of the BBB.

Glucose-dependent phosphorylation signaling pathways and crosstalk to mitochondrial respiration in insulin secreting cells.

BACKGROUND: Glucose is the main secretagogue of pancreatic beta-cells. Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation also initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways. Kinase substrate enrichment analysis (KSEA) was applied to identify key regulated kinases and phosphatases. Glucose-induced oxygen consumption was measured using a XF96 Seahorse instrument to reveal cross talk between glucose-regulated kinases and mitochondrial activation. RESULTS: Our kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Kinase-substrate enrichment identified upstream kinases and phosphatases and time-dependent activity changes during glucose stimulation. Activity trajectories of well-known glucose-regulated kinases and phosphatases are described. In addition, we predict activity changes in a number of kinases including NUAK1, not or only poorly studied in the context of the pancreatic beta-cell. Furthermore, we pharmacologically tested whether signaling pathways predicted by kinase-substrate enrichment analysis affected glucose-dependent acceleration of mitochondrial respiration. We find that phosphoinositide 3-kinase, Ca2+/calmodulin dependent protein kinase and protein kinase C contribute to short-term regulation of energy metabolism. CONCLUSIONS: Our results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.

Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

In pancreatic ß-cells, mitochondria generate signals that promote insulin granule exocytosis. Here we study how lysine acetylation of mitochondrial proteins mechanistically affects metabolism-secretion coupling in insulin-secreting cells. Using mass spectrometry-based proteomics, we identified lysine acetylation sites in rat insulinoma cell line clone 1E cells. In cells lacking the mitochondrial lysine deacetylase sirtuin-3 (SIRT3), several matrix proteins are hyperacetylated. Disruption of the SIRT3 gene has a deleterious effect on mitochondrial energy metabolism and Ca2+ signaling. Under resting conditions, SIRT3 deficient cells are overactivated, which elevates the respiratory rate and enhances calcium signaling and basal insulin secretion. In response to glucose, the SIRT3 knockout cells are unable to mount a sustained cytosolic ATP response. Calcium signaling is strongly reduced and the respiratory response as well as insulin secretion are blunted. We propose mitochondrial protein lysine acetylation as a control mechanism in ß-cell energy metabolism and Ca2+ signaling.-De Marchi, U., Galindo, A. N., Thevenet, J., Hermant, A., Bermont, F., Lassueur, S., Domingo, J. S., Kussmann, M., Dayon, L., Wiederkehr, A. Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

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

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

Nuclear Proteomics Uncovers Diurnal Regulatory Landscapes in Mouse Liver.

Diurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus.

High-throughput and simultaneous quantitative analysis of homocysteine-methionine cycle metabolites and co-factors in blood plasma and cerebrospinal fluid by isotope dilution LC-MS/MS.

The methionine cycle is a key pathway contributing to the regulation of human health, with well-established involvement in cardiovascular diseases and cognitive function. Changes in one-carbon cycle metabolites have also been associated with mild cognitive decline, vascular dementia, and Alzheimer's disease. Today, there is no single analytical method to monitor both metabolites and co-factors of the methionine cycle. To address this limitation, we here report for the first time a new method for the simultaneous quantitation of 17 metabolites in the methionine cycle, which are homocysteic acid, taurine, serine, cysteine, glycine, homocysteine, riboflavin, methionine, pyridoxine, cystathionine, pyridoxamine, S-adenosylhomocysteine, S-adenosylmethionine, betaine, choline, dimethylglycine, and 5-methyltetrahydrofolic acid. This multianalyte method, developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), provides a highly accurate and precise quantitation of these 17 metabolites for both plasma and cerebrospinal fluid metabolite monitoring. The method requires a simple sample preparation, which, combined with a short chromatographic run time, ensures a high sample throughput. This analytical strategy will thus provide a novel metabolomics approach to be employed in large-scale observational and intervention studies. We expect such a robust method to be particularly relevant for broad and deep molecular phenotyping of individuals in relation to their nutritional requirements, health monitoring, and disease risk management.