14-3-3ε Boosts Bleomycin-induced DNA Damage Response by Inhibiting the Drug-resistant Activity of MVP

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidence have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anticancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using an LC–MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein–protein interactions

Synthesis of Highly Water-Dispersible Polydopamine-Modified Multiwalled Carbon Nanotubes for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Analysis

In this work, we synthesized highly water-dispersible multiwalled carbon nanotubes@polydopamine (MWCNTs@PDA) core–shell composites by a facile in situ oxidative polymerization. The composites were successfully applied as a novel matrix for the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) analysis of various water-soluble small molecule compounds. It was found that MWCNTs@PDA composites have a higher sensitivity and peak intensities for small molecules detection

Designed Synthesis of Aptamer-Immobilized Magnetic Mesoporous Silica/Au Nanocomposites for Highly Selective Enrichment and Detection of Insulin

We designed and synthesized aptamer-immobilized magnetic mesoporous silica/Au nanocomposites (MMANs) for highly selective detection of unlabeled insulin in complex biological media using MALDI-TOF MS. The aptamer was easily anchored onto the gold nanoparticles in the mesochannels of MMANs with high capacity for highly efficient and specific enrichment of insulin. With the benefit from the size-exclusion effect of the mesoporous silica shell with a narrow pore size distribution (∼2.9 nm), insulin could be selectively detected despite interference from seven untargeted proteins with different size dimensions. This method exhibited an excellent response for insulin in the range 2–1000 ng mL^–1. Moreover, good recoveries in the detection of insulin in 20-fold diluted human serum were achieved. We anticipate that this novel method could be extended to other biomarkers of interest and potentially applied in disease diagnostics

Synthesis of Fe₃O₄/Graphene/TiO₂ Composites for the Highly Selective Enrichment of Phosphopeptides from Biological Samples

In this work, Fe₃O₄/graphene/TiO₂ composites with a large surface area were designed and synthesized for the selective extraction and enrichment of phosphopeptides from biological samples. First, magnetic graphene was prepared according to our previous method. Next, we made the Fe₃O₄/graphene/TiO₂ composite precursor using tetrabutyl titanate. Fe₃O₄/graphene/TiO₂ composites were obtained after solvothermal and calcination treatments. We used standard protein-digestion solutions and human liver samples to test the enrichment ability of the obtained Fe₃O₄/graphene/TiO₂ composites. The experimental results demonstrate that Fe₃O₄/graphene/TiO₂ composites have a good phosphopeptide enrichment ability

Multifunctional Nanoreactor for Comprehensive Characterization of Membrane Proteins Based on Surface Functionalized Mesoporous Foams

An integrated protocol is proposed here for efficient analysis of membrane proteins based on surface functionalized mesoporous graphene foams (MGF). The inherent hydrophobic nature of MGF and surface modification with hydrophilic chitosan (CS) make it highly suitable for the enrichment of hydrophobic membrane proteins from organic solvent, while remaining well-dispersed in aqueous solution for subsequent proteolysis. Therefore, such a multifunctional reactor ensures a facile solvent adjustment route. Furthermore, as a chitosan modified nanoporous reactor, it also provides a biocompatible nanoenvironment that can maintain the stability and activity of enzymes to realize efficient <i>in situ</i> digestion of the enriched membrane proteins. The concept was first proved with a standard hydrophobic membrane protein, bacteriorhodopsin, where a high number of identified peptides and amino acid sequence coverage were achieved even at extremely low protein concentration. The mesoporous reaction system was further applied to the analysis of complex real-case proteome samples, where 931 membrane proteins were identified in triplicate analyses by 2D LC-MS/MS. In contrast, with in-solution proteolysis, only 73 membrane proteins were identified from the same sample by the same 2D LC-MS/MS. The identified membrane proteins by the MGF-CS protocol include many biomarkers of the cell line. These results suggest that the multifunctional MGF-CS protocol is of great value to facilitate the comprehensive characterization of membrane proteins in the proteome research

14-3-3ε Mediates the Cell Fate Decision-Making Pathways in Response of Hepatocellular Carcinoma to Bleomycin-Induced DNA Damage

<div><p>Background</p><p>Lack of understanding of the response of hepatocellular carcinoma (HCC) to anticancer drugs causes the high mortality of HCC patients. Bleomycin (BLM) that induces DNA damage is clinically used for cancer therapy, while the mechanism underlying BLM-induced DNA damage response (DDR) in HCC cells remains ambiguous. Given that 14-3-3 proteins are broadly involved in regulation of diverse biological processes (BPs)/pathways, we investigate how a 14-3-3 isoform coordinates particular BPs/pathways in BLM-induced DDR in HCC.</p> <p>Methodology/Principal Findings</p><p>Using dual-tagging quantitative proteomic approach, we dissected the 14-3-3ε interactome formed during BLM-induced DDR, which revealed that 14-3-3ε <i>via</i> its associations with multiple pathway-specific proteins coordinates multiple pathways including chromosome remodeling, DNA/RNA binding/processing, DNA repair, protein ubiquitination/degradation, cell cycle arrest, signal transduction and apoptosis. Further, “zoom-in” investigation of the 14-3-3ε interacting network indicated that the BLM-induced interaction between 14-3-3ε and a MAP kinase TAK1 plays a critical role in determining cell propensity of apoptosis. Functional characterization of this interaction further revealed that BLM triggers site-specific phosphorylations in the kinase domain of TAK1. These BLM-induced changes of phosphorylations directly correlate to the strength of the TAK1 binding to 14-3-3ε, which govern the phosphorylation-dependent TAK1 activation. The enhanced 14-3-3ε-TAK1 association then inhibits the anti-apoptotic activity of TAK1, which ultimately promotes BLM-induced apoptosis in HCC cells. In a data-dependent manner, we then derived a mechanistic model where 14-3-3ε plays the pivotal role in integrating diverse biological pathways for cellular DDR to BLM in HCC.</p> <p>Conclusions</p><p>Our data demonstrated on a systems view that 14-3-3ε coordinates multiple biological pathways involved in BLM-induced DDR in HCC cells. Specifically, 14-3-3ε associates with TAK1 in a phosphorylation-dependent manner to determine the cell fate of BLM-treated HCC cells. Not only individual proteins but also those critical links in the network could be the potential targets for BLM-mediated therapeutic intervention of HCC.</p> </div

Multilayer Hydrophilic Poly(phenol-formaldehyde resin)-Coated Magnetic Graphene for Boronic Acid Immobilization as a Novel Matrix for Glycoproteome Analysis

Capturing glycopeptides selectively and efficiently from mixed biological samples has always been critical for comprehensive and in-depth glycoproteomics analysis, but the lack of materials with superior capture capacity and high specificity still makes it a challenge. In this work, we introduce a way first to synthesize a novel boronic-acid-functionalized magnetic graphene@phenolic-formaldehyde resin multilayer composites via a facile process. The as-prepared composites gathered excellent characters of large specific surface area and strong magnetic responsiveness of magnetic graphene, biocompatibility of resin, and enhanced affinity properties of boronic acid. Furthermore, the functional graphene composites were shown to have low detection limit (1 fmol) and good selectivity, even when the background nonglycopeptides has a concentration 100 fold higher. Additionally, enrichment efficiency of the composites was still retained after being used repeatedly (at least three times). Better yet, the practical applicability of this approach was evaluated by the enrichment of human serum with a low sample volume of 1 μL. All the results have illustrated that the magG@PF@APB has a great potential in glycoproteome analysis of complex biological samples

Simultaneous Online Enrichment and Identification of Trace Species Based on Microfluidic Droplets

A facile online enrichment protocol has been proposed based on microfluidic droplets acting as an interface between a liquid chromatography separation system and detection systems of ESI-MS/MS and laser-induced fluorescence. Low-abundance species were successfully concentrated and analyzed in this system via droplet shrinkage. The proposed platform significantly increased the enrichment efficiency and detection sensitivity with reduced sample handling steps, short analysis time, and no cross-contamination. The presented system is universal, shows no discrimination, and is easily coupled with other separation and detection approaches

Mechanistic model underlying how 14-3-3ε integrates multiple biological processes (BPs) to coordinate BLM-induced DDR.

<p>The red arrow from 14-3-3ε towards specific proteins indicates our validated BLM-induced 14-3-3ε interactors. The components including SMARCC2/ MTA2/RBBP4<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055268#pone.0055268-Zhang1" target="_blank">[41]</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055268#pone.0055268-Battaglioli1" target="_blank">[43]</a>, MTAR3/SFPQ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055268#pone.0055268-Salton1" target="_blank">[46]</a>, and TAB3 (MAP3K7IP3) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055268#pone.0055268-Cheung1" target="_blank">[51]</a> that were previously reported to form complexes with HDAC1, NONO and TAK1 respectively were also indentified by MS.</p

Isolation of 14-3-3ε complex by immunoprecipitation.

<p>Purification of 14-3-3ε complex was carried out via the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055268#s4" target="_blank">Materials and Methods</a>. Proteins were separated on SDS-PAGE and stained with Coomassie Blue. WCL: whole cell lysates; IP: immunoprecipitates; MK: protein marker. Bait protein FLAG-tagged 14-3-3ε is indicated by arrow on SDS-PAGE gel.</p