Taurodeoxycholate Increases the Number of Myeloid-Derived Suppressor Cells That Ameliorate Sepsis in Mice.

Bile acids (BAs) control metabolism and inflammation by interacting with several receptors. Here, we report that intravenous infusion of taurodeoxycholate (TDCA) decreases serum pro-inflammatory cytokines, normalizes hypotension, protects against renal injury, and prolongs mouse survival during sepsis. TDCA increases the number of granulocytic myeloid-derived suppressor cells (MDSCLT) distinctive from MDSCs obtained without TDCA treatment (MDSCL) in the spleen of septic mice. FACS-sorted MDSCLT cells suppress T-cell proliferation and confer protection against sepsis when adoptively transferred better than MDSCL. Proteogenomic analysis indicated that TDCA controls chromatin silencing, alternative splicing, and translation of the immune proteome of MDSCLT, which increases the expression of anti-inflammatory molecules such as oncostatin, lactoferrin and CD244. TDCA also decreases the expression of pro-inflammatory molecules such as neutrophil elastase. These findings suggest that TDCA globally edits the proteome to increase the number of MDSCLT cells and affect their immune-regulatory functions to resolve systemic inflammation during sepsis.

Low serum Kallistatin level was associated with poor neurological outcome of out-of-hospital cardiac arrest survivors: Proteomics study.

AIM OF THE STUDY: To identify proteins of which depletion are associated with the poor 6-month neurological outcome of out-of-hospital cardiac arrest survivors. METHODS: Seven healthy volunteers and 34 out-of-hospital cardiac arrest survivors admitted to the intensive care unit (ICU) and underwent targeted-temperature management were enrolled. According to the 6-month cerebral performance category (CPC) scale, patients were divided into the good (CPC 1-2) and poor (CPC 3-5) outcome groups. Blood samples were obtained at 0, 24, and 72 h after admission to the ICU. RESULTS: With proteomic approaches, we found 23 proteins that showed group-differences between the sera pooled from 7 study groups: healthy volunteers, the good outcome groups (0, 24, and 72 h), and the poor outcome groups (0, 24, and 72 h). We selected 7 candidate proteins of which intensities were different between the good and poor outcome groups (>2-fold change) and excluded 5 proteins related to haemolysis or remaining high abundant proteins. To confirm the 2 identified proteins: retinal dehydrogenase 1 and Kallistatin, we performed enzyme-linked immunosorbent assay with individual serum. Finally, old age (odds ratio = 1.055; 95% confidence interval, 1.002-1.112; p = 0.043) and low serum kallistatin level at 0 h (odds ratio = 0.784; 95% confidence interval, 0.618-0.995; p = 0.046) were independently associated with the poor 6-month neurological outcome. CONCLUSION: The depletion of serum kallistatin at admission to the ICU was associated with the poor neurological outcome of out-of-hospital cardiac arrest survivors.

Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -ß1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry.

The heterotrimeric enzyme AMP-activated protein kinase (AMPK) is a major metabolic factor that regulates the homeostasis of cellular energy. In particular, AMPK mediates the insulin resistance that is associated with type 2 diabetes. Generally, cellular processes require tight regulation of protein kinases, which is effected through their formation of complex with other proteins and substrates. Despite their critical function in regulation and pathogenesis, there are limited data on the interaction of protein kinases. To identify proteins that interact with AMPK, we performed large-scale affinity purification (AP)-mass spectrometry (MS) of the AMPK-α1 and -ß1 subunits. Through a comprehensive analysis, using a combination of immunoprecipitaion and ion trap mass spectrometry, we identified 381 unique proteins in the AMPKα/ß interactomes: 325 partners of AMPK-α1 and 243 for AMPK-ß1. Further, we identified 196 novel protein-protein interactions with AMPK-α1 and AMPK-ß1. Notably, in our bioinformatics analysis, the novel interaction partners mediated functions that are related to the regulation of actin organization. Specifically, several such proteins were linked to pancreatic beta-cell functions, including glucose-stimulated insulin secretion, beta-cell development, beta-cell differentiation, and cell-cell communication.

Characterization of the membrane proteome and N-glycoproteome in BV-2 mouse microglia by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Microglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes. In particular, microglial activation and communication between microglia, astrocytes, and neurons are hallmarks of the pathogenesis of several neurodegenerative diseases. Membrane proteins and their N-linked glycosylation mediate this microglial activation and regulate many biological process including signal transduction, cell-cell communication, and the immune response. Although membrane proteins and N-glycosylation represent a valuable source of drug target and biomarker discovery, the knowledge of their expressed proteome in microglia is very limited. RESULTS: To generate a large-scale repository, we constructed a membrane proteome and N-glycoproteome from BV-2 mouse microglia using a novel integrated approach, comprising of crude membrane fractionation, multienzyme-digestion FASP, N-glyco-FASP, and various mass spectrometry. We identified 6928 proteins including 2850 membrane proteins and 1450 distinct N-glycosylation sites on 760 N-glycoproteins, of which 556 were considered novel N-glycosylation sites. Especially, a total of 114 CD antigens are identified via MS-based analysis in normal conditions of microglia for the first time. Our bioinformatics analysis provides a rich proteomic resource for examining microglial function in, for example, cell-to-cell communication and immune responses. CONCLUSIONS: Herein, we introduce a novel integrated proteomic approach for improved identification of membrane protein and N-glycosylation sites. To our knowledge, this workflow helped us to obtain the first and the largest membrane proteomic and N-glycoproteomic datesets for mouse microglia. Collectively, our proteomics and bioinformatics analysis significantly expands the knowledge of the membrane proteome and N-glycoproteome expressed in microglia within the brain and constitutes a foundation for ongoing proteomic studies and drug development for various neurological diseases.

In-depth proteomic analysis of mouse microglia using a combination of FASP and StageTip-based, high pH, reversed-phase fractionation.

Microglia are major immune cells in the central nervous system. A characterization of microglia proteome would facilitate on the study of microglial functions in association with various neurodegenerative diseases. To build a reference proteome, we established a BV-2 microglial proteome to a depth of 5494 unique protein groups using a novel strategy that combined FASP, StageTip-based high pH fractionation, and high-resolution MS quickly and cost efficiently. By bioinformatics analysis, the BV-2 proteome is a valuable resource for studies of microglial function, such as in the immune response, inflammatory response, and phagocytosis. All MS data have been deposited in the ProteomeXchange with identifier PXD000168.

Comprehensive phosphoproteome analysis of INS-1 pancreatic ß-cells using various digestion strategies coupled with liquid chromatography-tandem mass spectrometry.

Type 2 diabetes results from aberrant regulation of the phosphorylation cascade in beta-cells. Phosphorylation in pancreatic beta-cells has not been examined extensively, except with regard to subcellular phosphoproteomes using mitochondria. Thus, robust, comprehensive analytical strategies are needed to characterize the many phosphorylated proteins that exist, because of their low abundance, the low stoichiometry of phosphorylation, and the dynamic regulation of phosphoproteins. In this study, we attempted to generate data on a large-scale phosphoproteome from the INS-1 rat pancreatic beta-cell line using linear ion trap MS/MS. To profile the phosphoproteome in-depth, we used comprehensive phosphoproteomic strategies, including detergent-based protein extraction (SDS and SDC), differential sample preparation (in-gel, in-solution digestion, and FASP), TiO2 enrichment, and MS replicate analyses (MS2-only and multiple-stage activation). All spectra were processed and validated by stringent multiple filtering using target and decoy databases. We identified 2467 distinct phosphorylation sites on 1419 phosphoproteins using 4 mg of INS-1 cell lysate in 24 LC-MS/MS runs, of which 683 (27.7%) were considered novel phosphorylation sites that have not been characterized in human, mouse, or rat homologues. Our informatics data constitute a rich bioinformatics resource for investigating the function of reversible phosphorylation in pancreatic beta-cells. In particular, novel phosphorylation sites on proteins that mediate the pathology of type 2 diabetes, such as Pdx-1, Nkx.2, and Srebf1, will be valuable targets in ongoing phosphoproteomics studies.

Detection of differential proteomes associated with the development of type 2 diabetes in the Zucker rat model using the iTRAQ technique.

Type 2 diabetes (T2D) is closely associated with obesity, and it arises when pancreatic ß cells fail to achieve ß cell compensation. However, the mechanism linking obesity, insulin resistance, and ß cell failure in T2D is not fully understood. To explore this association, we carried out a differential proteomics study using the disease models of Zucker Fatty (ZF) and Zucker Diabetic Fatty (ZDF) rats as the rat models for obese/prediabetes and obese/diabetes, respectively. Differentially expressed islet proteins were identified among ZDF, ZF, and Zucker Lean (ZL, control rat) rats using three iTRAQ experiments, where three biological replicates and two technical replicates were examined to assess both the technical and biological reproducibilities. A total of 54 and 58 proteins were differentially expressed in ZDF versus ZL rats and in ZF versus ZL rats, respectively. Notably, the novel proteins involved in impaired insulin secretion (Scg2, Anxa2, and Rab10), mitochondrial dysfunction (Atp5b and Atp5l), extracellular matrix proteins (Lgal-1, Vim, and Fbn1), and microvascular ischemia (CPA1, CPA2, CPB, Cela2a, and Cela3b) were observed for the first time. With these novel proteins, our proteomics study could provide valuable clues for better understanding the underlying mechanisms associated with the dynamic transition of obesity to T2D.