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1.
Talanta ; 207: 120340, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594582

RESUMO

The emitter clogging is the most common hardware failure of nano-electrospray ionization, to improve the durability and electrospray stability of fused silica emitters, we demonstrate a means of fabricating nano-electrospray emitters with controllable aperture size and gradually-narrowed channel on the tip. We simulated the fluid morphologies in the emitter channels by computational fluid dynamics and found more stable flow on aperture-controllable nano-electrospray emitter. Besides, we found the unstable flow sections of commercial emitters match the actual clogging sections very well, indicating the main cause of emitter clogging is unstable flow. We further tested the emitters by nano-LC-MS based proteome analysis. Compared with the commercial emitter, aperture-controllable nano-electrospray emitters promoted the total ion chromatogram intensity by 25%, the number of identified proteins by 6.58%, and the number of identified peptides by 7.87%. In total, 989 proteins were identified from 1 µg of extracted mouse cardiac proteins. After the optimization by using mouse samples, we analyzed clinical auricular dextral tissues from patients undergoing cardiac surgery and found 16 proteins related to atrial fibrillation. Overall, aperture-controllable nano-electrospray emitter exhibits better sensitivity and reproducibility in the application of nano-LC-MS cardiac proteome analysis.

2.
Anal Chem ; 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31751117

RESUMO

Protein N-glycosylation is ubiquitous in the brain and is closely related to cognition and memory. Alzheimer's disease (AD) is a multifactorial disorder that lacks a clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. However, the systematic variations in protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technical challenges. Here, we applied multilayered N-glycoproteomics to quantify the global protein expres-sion levels, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD (APP/PS1 transgenic) and wild-type mouse brains. The N-glycoproteomic landscape exhibited highly complex site-specific heterogeneity in AD mouse brains. The generally dysregulated N-glycosylation in AD, which involved proteins such as glutamate receptors as well as fucosylated and oligomannose glycans, were explored by quantitative analyses. Furthermore, a functional study revealed the crucial effects of N-glycosylation on proteins and neurons. Our work provides a systematic multilayered N-glycoproteomic strategy for AD and can be applied to di-verse biological systems.

3.
Anal Chem ; 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31775502

RESUMO

Histone modifications play an important role in regulating transcriptional gene expression and chromatin processes in eukaryotes. Increasing researches proved that aberrant post-translational modifications (PTMs) of histones is associated with many diseases. However, MS-based identification and quantification histone PTMs are still challenging. Although classic chemical derivatization in conjunction with trypsin digestion is a widely used for histone PTMs analysis in bottom-up strategy, several side reactions have been observed in practice. In this work, outer membrane protease T (OmpT) was utilized as a protease for direct histone proteolysis and generated appropriate lengths of histone peptide for retention on reversed-phase chromatography. The powerful and unique tolerance of OmpT for modified lysines and arginines was demonstrated and can be quantitatively described for the first time, making it useful for detecting natural modifications. Using the optimized digestion conditions, we succeeded to identify 121 histone marks from HEK293T cells, 42 of which were previously unreported. Additionally, histone H3 PTMs were quantitatively profiled in the KMS11 multiple myeloma cells and NSD2 selective knockout KMS11cells, revealed the NSD2 was of high specificity on H3K36 dimethylation. Histone chemical derivatizations are not required in our strategy, showing a remarkable strength over the conventional trypsin-based workflow.

4.
FASEB J ; 33(11): 13040-13050, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31487196

RESUMO

Embryonic stem cells (ESCs) are pluripotent stem cells with the ability to self-renew and to differentiate into any cell types of the 3 germ layers. Recent studies have demonstrated that there is a strong connection between mitochondrial function and pluripotency. Here, we report that methyltransferase like (Mettl) 17, identified from the clustered regularly interspaced short palindromic repeats knockout screen, is required for proper differentiation of mouse embryonic stem cells (mESCs). Mettl17 is located in mitochondria through its N-terminal targeting sequence and specifically interacts with 12S mitochondrial ribosomal RNA (mt-rRNA) as well as small subunits of mitochondrial ribosome (MSSUs). Loss of Mettl17 affects the stability of both 12S mt-rRNA and its associated proteins of MSSUs. We further showed that Mettl17 is an S-adenosyl methionine (SAM)-binding protein and regulates mitochondrial ribosome function in a SAM-binding-dependent manner. Loss of Mettl17 leads to around 70% reduction of m4C840 and 50% reduction of m5C842 of 12S mt-rRNA, revealing the first regulator of the m4C840 and indicating a crosstalk between the 2 nearby modifications. The defects of mitochondrial ribosome caused by deletion of Mettl17 lead to the impaired translation of mitochondrial protein-coding genes, resulting in significant changes in mitochondrial oxidative phosphorylation and cellular metabolome, which are important for mESC pluripotency.-Shi, Z., Xu, S., Xing, S., Yao, K., Zhang, L., Xue, L., Zhou, P., Wang, M., Yan, G., Yang, P., Liu, J., Hu, Z., Lan, F. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes.

5.
Hepatology ; 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31539182

RESUMO

Hepatocellular carcinoma (HCC) often accompanies with resistance to immunotherapies despite the presence of tumor-infiltrating lymphocytes. Here we report that HDAC6 represses TH 17 cell pathogenicity and antitumor immune response dependent on its deacetylase activity. Adoptive transfer of HDAC6-deficient TH 17 cell impedes HCC growth dependent on elevated IL-17A via enhancing the production of antitumor cytokine and CD8+ T cell-mediated antitumor responses. Intriguingly, HDAC6-depleted T cells triggers PD-1-PD-L1 expression to achieve a strong synergistic effect to sensitize advanced HCC to immune checkpoint blocker, while blockade of IL-17A partially suppresses it. Mechanistically, HDAC6 limits TH 17 pathogenicity and antitumor effect through regulating FoxO1. HDAC6 binds and deacetylates cytosolic FoxO1 at K242, which is required for its nuclear translocation and stabilization to repress RoRγt, the transcription factor of TH 17 cell. This regulation of HDAC6 for murine and human TH 17 cell is highly conserved. CONCLUSION: These results demonstrate targeting cytosolic HDAC6-FoxO1 axis reprograms the pathogenicity and antitumor response of TH 17 cells in HCC, with a pathogenicity-driven responsiveness to facilitate immunotherapies.

6.
Anal Chim Acta ; 1080: 116-126, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409460

RESUMO

Histones participate in epigenetic regulation via dynamic post-translational modifications (PTMs) of histone variants. Comprehensive characterization of histone markers, especially those for the histone variants, could help to decipher the mechanism of epigenetic regulation. However, correctly profiling histone PTMs and its variants using mass spectrometry remains a challenge. Here, we developed an improved, specific and sensitive LC separation in conjunction with a high throughput multiple reaction monitoring combined with stable isotope-labeled internal standards (MRM-SIS) based quantitative method for histone H3 variants in the study of epigenetic regulation in the cell cycle. PTM patterns and the overall abundance of the three main histone H3 variants from Karpas 422 cells were analyzed and quantified simultaneously during different cell stages. The methylation pattern varied between different sites and modification states during the cell cycle. The canonical H3.1 presented regular patterns on K27 and K36, similar to H3.2, albeit differing from variant H3.3. H3.3 K36me2 increased from G1, S to G2 phase, whereas the same marker decreased in both H3.1 and H3.2. This novel discovery inspires more focus and research on the histone variants behavior and function during cell cycle. Moreover, this improved method could be applied to unveil PTMs dynamics of histone variants in several biological processes.

7.
Anal Chem ; 91(19): 12435-12443, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31453685

RESUMO

Efficient detection of aberrant glycoproteins in serum is particularly important for biomarker discovery. However, direct quantitation of glycoproteins in serum remains technically challenging because of the extraordinary complexity of the serum proteome. In the current work, we proposed a straightforward and highly efficient strategy by using the nonglycopeptides releasing from the specifically enriched glycoproteins for targeted glycoprotein quantification. With this so-called nonglycopeptide-based mass spectrometry (NGP-MS) strategy, a powerful and nondiscriminatory pipeline for hepatocellular carcinoma (HCC) glycoprotein biomarker discovery, verification, and validation has been developed. First, a data set of 234 NGPs was strictly established for multiple-reaction monitoring (MRM) quantification in serum. Second, the NGPs enriched from 20 HCC serum mixtures and 20 normal serum mixtures were labeled with mTRAQ reagents (Δ0 and Δ8, respectively) to find the differentially expressed glycoproteins in HCC. A total of 97 glycoprotein candidates were preliminarily screened and submitted for absolute quantitation with NGP-based stable-isotope-labeled (SID)-MRM in the individual samples of 38 HCC serum and 24 normal controls. Finally, 21 glycoproteins were absolutely quantified with high quality. The diagnostic sensitivity results showed that three glycoproteins, ß-2-glycoprotein 1 (APOH), α-1-acid glycoprotein 2 (ORM2), and complement C3 (C3), could be used for the discrimination between HCC patients and healthy people. A novel glycoprotein biomarker panel [APOH, ORM2, C3, and α-fetoprotein (AFP)] has proven to outperform AFP, the known HCC serum biomarker, alone, in this study. We believe that this strategy and the panel of glycoproteins might hold great clinical value for HCC detection in the future.

8.
Talanta ; 204: 670-676, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357351

RESUMO

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4µ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4µ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol ß-casein, the 4µ-PEO-Ti4+ performed the best property with starting material of 99-132 µg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4µ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4µ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of ß-casein from a mixture with BSA of 1000-fold abundant. The 4µ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4µ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4µ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.

9.
Nat Commun ; 10(1): 3391, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358770

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome that elevates the risk of hepatocellular carcinoma (HCC). Although alteration of lipid metabolism has been increasingly recognized as a hallmark of cancer cells, the deregulated metabolic modulation of HCC cells in the NAFLD progression remains obscure. Here, we discovers an endoplasmic reticulum-residential protein, Nogo-B, as a highly expressed metabolic modulator in both murine and human NAFLD-associated HCCs, which accelerates high-fat, high-carbohydrate diet-induced metabolic dysfunction and tumorigenicity. Mechanistically, CD36-mediated oxLDL uptake triggers CEBPß expression to directly upregulate Nogo-B, which interacts with ATG5 to promote lipophagy leading to lysophosphatidic acid-enhanced YAP oncogenic activity. This CD36-Nogo-B-YAP pathway consequently reprograms oxLDL metabolism and induces carcinogenetic signaling for NAFLD-associated HCCs. Targeting the Nogo-B pathway may represent a therapeutic strategy for HCC arising from the metabolic syndrome.

10.
Talanta ; 199: 254-261, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952254

RESUMO

N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high-throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples. In this study, we firstly optimized traditional zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) enrichment and found that using of 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) as the enrichment buffer, 2800 g as the washing speed and 600 µL as the washing volume achieved the best specificity, which is higher than 75%. On this basis, we developed a multi-parallel enrichment strategy assisted by a filter-coated 96-well plate, which achieved high specificity and high throughput simultaneously. This strategy allowed us to enrich large numbers of fractionated samples from hepatocellular carcinoma (HCC) cell lines in less than 2 h. Its good specificity helped us achieve in-depth mapping of the N-glycoproteome in metastatic HCC cell lines. A total of 5466 N-glycosites from 2383 glycoproteins were identified, among which 1900 N-glycosites were unannotated in UniProt. The in-depth glycoproteome mapping provides insight into the N-glycosylation status in HCC cell lines with differences in metastatic potential and contributes to biomarker discovery.


Assuntos
Carcinoma Hepatocelular/química , Glicopeptídeos/química , Ensaios de Triagem em Larga Escala , Neoplasias Hepáticas/química , Proteoma/análise , Carcinoma Hepatocelular/metabolismo , Cromatografia Líquida , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Neoplasias Hepáticas/metabolismo , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Células Tumorais Cultivadas
11.
Cancer Genomics Proteomics ; 16(1): 81-89, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30587502

RESUMO

BACKGROUND/AIM: Pancreatic cancer (PC) is currently the fourth leading cause of cancer-related mortality worldwide. Peripheral blood mononuclear cells (PBMCs) is a subpopulation of accessible and functional immune cells. Comparative analysis of the proteome of PBMCs can help us elucidate the mechanism of disease and find potential biomarkers for diagnosis. MATERIALS AND METHODS: PBMCs were collected from healthy individuals, patients with benign diseases, and pancreatic cancer. iTRAQ-2DLC-MS/MS and SWATH methodologies were applied to make a comparative proteomics analysis of PBMCs. RESULTS: A total of 3,357 proteins with a false discovery rate (FDR) <1% were identified, of which 114 proteins were found dysregulated in the PC group. An extensive SWATH library was constructed which showed a potential application for large scale clinical sample analysis. CONCLUSION: A PBMCs proteome with extensive protein representation was achieved, which will potentially allow the identification of novel biomarkers for PC.


Assuntos
Biomarcadores Tumorais , Leucócitos Mononucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteoma , Proteômica , Cromatografia Líquida , Biologia Computacional/métodos , Curadoria de Dados , Redes Reguladoras de Genes , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Espectrometria de Massas em Tandem
12.
Nat Commun ; 9(1): 5214, 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30523261

RESUMO

Obesity increases the risk of hepatocellular carcinoma (HCC) especially in men, but the molecular mechanism remains obscure. Here, we show that an androgen receptor (AR)-driven oncogene, cell cycle-related kinase (CCRK), collaborates with obesity-induced pro-inflammatory signaling to promote non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Lentivirus-mediated Ccrk ablation in liver of male mice fed with high-fat high-carbohydrate diet abrogates not only obesity-associated lipid accumulation, glucose intolerance and insulin resistance, but also HCC development. Mechanistically, CCRK fuels a feedforward loop by inducing STAT3-AR promoter co-occupancy and transcriptional up-regulation, which in turn activates mTORC1/4E-BP1/S6K/SREBP1 cascades via GSK3ß phosphorylation. Moreover, hepatic CCRK induction in transgenic mice stimulates mTORC1-dependent G-csf expression to enhance polymorphonuclear myeloid-derived suppressor cell recruitment and tumorigenicity. Finally, the STAT3-AR-CCRK-mTORC1 pathway components are concordantly over-expressed in human NASH-associated HCCs. These findings unveil the dual roles of an inflammatory-CCRK circuitry in driving metabolic and immunosuppressive reprogramming through mTORC1 activation, thereby establishing a pro-tumorigenic microenvironment for HCC development.

13.
Proc Natl Acad Sci U S A ; 115(38): E8863-E8872, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30190427

RESUMO

Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Fosfotirosina/metabolismo , Engenharia de Proteínas/métodos , Proteômica/métodos , Animais , Benzimidazóis/farmacologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Espectrometria de Massas , Camundongos Transgênicos , Fosforilação , Piperidinas/farmacologia , Ligação Proteica , Receptor ErbB-2/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Raios Ultravioleta , Domínios de Homologia de src/genética , Domínios de Homologia de src/efeitos da radiação
14.
Anal Chem ; 90(17): 10394-10399, 2018 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-30075082

RESUMO

Single-cell analysis provides an important strategy to evaluate cellular heterogeneity. Although surface-enhanced Raman scattering (SERS) has been considered as a promising label-free technique for single-cell analysis, it remains at the early stage for characterizing the extracellular metabolites of single cells. Herein, we developed a convenient, flexible, and straightforward three-dimensional (3D) plasmonic trap array for simultaneously compartmentalizing and sensitively detecting single-cell metabolites. The 3D trap was spontaneously self-formed by an interfacial-energy-driven process when a liquid droplet was covered with an immiscible oil liquid (polydimethylsiloxane, PDMS). When a droplet of pure AgNO3 solution was immersed into PDMS, Ag+ ions were automatically reduced by the residual Si-H groups in PDMS. Snowflake-like nanoparticles of Ag could be formed on the inner surface of the 3D traps by tuning the concentration of Ag salt precursors and then assembled to flower-like microstructures, endowing the traps with remarkable plasmon enhancement. The established 3D traps exhibited considerably enhanced surface plasmon resonance signals for Raman reporting, and a low detection limit at the aM level was achieved for p-aminothiophenol. Moreover, these 3D traps can serve as an efficient tool for single-cell SERS measurement. As a proof-of-concept, dipicolinic acid, a common biomarker of bacterial spores, was successfully detected from a single cell. The presented approach provides a versatile tool for label-free and sensitive detection of single-cell environments.

15.
Chem Sci ; 9(29): 6271-6285, 2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30123482

RESUMO

Given that unnatural sugar expression is metabolically achieved, the kinetics and disposition of incorporation can lend insight into the temporal and localization preferences of sialylation across the cell surface. However, common detection schemes lack the ability to detail the molecular diversity and distribution of target moieties. Here we employed a mass spectrometric approach to trace the placement of azido sialic acids on membrane glycoconjugates, which revealed substantial variations in incorporation efficiencies between N-/O-glycans, glycosites, and glycosphingolipids. To further explore the propensity for sialylation, we subsequently mapped the native glycome of model epithelial cell surfaces and illustrate that while glycosylation sites span broadly across the extracellular region, a higher number of heterogeneous glycoforms occur on sialylated sites closest to the transmembrane domain. Beyond imaging techniques, this integrative approach provides unprecedented details about the frequency and structure-specific distribution of cell surface sialylation, a critical feature that regulates cellular interactions and homeostatic pathways.

16.
Analyst ; 143(19): 4526-4536, 2018 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-30128447

RESUMO

There are thousands of lipid species existing in cells, which belong to eight different categories. Lipids are the essential building blocks of cells. Recent studies have started to unveil the important functions of lipids in regulating cell metabolism. However, we are still at a very early stage in fully understanding the physiological and pathological functions of lipids. The application of lipidomics for studying lipid metabolism can provide a direct readout of the cellular status and broadens our understanding of the mechanisms that underpin metabolic disease states. This review provides an introduction to lipid metabolism and its role in modulating homeostasis and immunity. We also describe representative applications of lipidomics for studying lipid metabolism in inflammation-related diseases.

17.
Cell Discov ; 4: 52, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30131868

RESUMO

Pericardial adipose tissue, which comprises both epicardial adipose tissue (EAT) and paracardial adipose tissue (PAT), has recently been recognized as a novel factor in the pathophysiology of cardiovascular diseases, especially coronary artery disease (CAD). The goal of this study was to evaluate differences in the brown-like characteristic and proteome among human EAT, PAT, and subcutaneous adipose tissue (SAT) to identify candidate molecules causing CAD. Uncoupling protein 1 (UCP-1) and other brown-related proteins were highly expressed in pericardial adipose tissue but was weakly expressed in SAT from the same non-CAD patient. Moreover, pericardial adipose tissues displayed a higher thermogenesis than SAT. However, brown-related genes were lower in CAD pericardial fat. Remarkably, there were lower levels of metabolic enzymes involved in glycolysis, tricarboxylic acid cycle, and fatty acid metabolism in pericardial adipose tissues of CAD. EAT is an organ adjacent to aortic root without anatomy barriers, which differs from PAT. We found that the expression of ribosomal protein S3A (RPS3A) was decreased in human EAT as well as in mouse perivascular adipose tissue (PVAT). Knockdown of RPS3A significantly inhibited adipocyte differentiation in preadipocytes and impaired the function of mitochondria in mature adipocytes. Moreover, RPS3A knockdown in mouse periaortic adipose tissue impaired browning of PVAT, accelerated vascular inflammation, and atherosclerosis progression. Mechanistically, RPS3A can migrate to the mitochondria to maintain the function of brown adipocytes. These findings provide compelling evidence that RPS3A was a key factor for modulating the brown fat-specific gene UCP-1 and carbon metabolic enzymes in EAT for preventing CAD.

18.
Nature ; 559(7715): 637-641, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30022161

RESUMO

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.

19.
Anal Chem ; 90(12): 7357-7363, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29851350

RESUMO

Protein glycosylation is a significant participant in a mass of biological processes, which is a pivotal protein post-translational modification. Due to the low contents of glycopeptides compared with nonglycopeptides and the microheterogeneity of glycosylation sites, highly selective enrichment methods for the purification of glycopeptides are required for the comprehensive characterization of glycoproteomics. In this work, a type of magnetic mesoporous phenolic resin (MMP) was prepared using branched polyethylenimine (PEI) as a cross-linker from a homogeneous magnetic Fe3O4@SiO2 solution in a resorcinol/formaldehyde monomer aqueous system via an in situ emulsion polymerization procedure. The results showed that MMP exhibited good biocompatibility, a mesoporous structure, nitrogen-containing functionality, excellent hydrophilicity, and solvent resistance by using multiple characterization methods. By taking advantage of the interaction between hydrophilic groups on the MMP and glycan components on the glycopeptides, the acquired MMP was utilized to the selective capture of N-glycopeptides (human IgG or HRP tryptic digests/BSA proteins = 1:50), good recovery yield (70.18-97.23%), superior binding capacity (400 mg g-1), and excellent reproducibility. Based on the outstanding performance in standard glycoproteins tryptic digests enrichment, MMP was further used to capture N-glycopeptides from tryptic digests of human serum. A total of 15 unique N-glycopeptides were identified from an ultralow sample volume (0.025 µL) of human serum. Overall, we identified 356 unique N-glycopeptides corresponding to 119 glycoproteins from human serum (0.35 µL) in the overlap of three replicate analyses. All the results have demonstrated that MMP has great potential in large-scale N-glycoproteomics research.

20.
Anal Chem ; 90(10): 6161-6168, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29660268

RESUMO

Palmitoylation, a type of fatty acylation, has vital roles in many biological processes. For ultradeep identification of protein palmitoylation, an enrichment approach based on a novel magnetic microsphere modified with 2,2'-dithiodipyridine (Fe3O4/SiO2-SSPy microsphere) is presented in this study. The Fe3O4/SiO2-SSPy microspheres were synthesized by directly coating thiol-containing silane coupling agent onto the magnetic supraparticles in aqueous solution at room temperature. Due to the intrinsic magnetic properties, high surface-to-volume ratios, and abundant reactive functional groups on the surface, these microspheres enabled direct capture of palmitoylated targets and convenient isolation, contributing to remarkable enrichment selectivity (purifying palmitoylated peptides from mixtures with nonpalmitoylated peptides even at a 1:500 molar ratio) and sensitivity (the detection limit was at femtomole level), thus enabling a global annotation of protein palmitoylation for complex biological samples. We successfully identified 1304 putative palmitoylated proteins from mouse brain tissues by using this method, which is the largest mouse palmitoylome data set to date. Except for those known members, many new proteins and pathways were also found to be regulated by palmitoylation.

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