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1.
J Proteomics ; 215: 103669, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-31987925

RESUMO

The selection of a data processing method for use in mass spectrometry-based label-free proteome quantification contributes significantly to its accuracy and precision. In this study, we comprehensively evaluated 7 commonly-used label-free quantification methods (MaxQuant-Spectrum count, MaxQuant-iBAQ, MaxQuant-LFQ, MaxQuant-LFAQ, Proteome Discoverer, MetaMorpheus, TPP-StPeter) with a focus on missing values, precision, accuracy, selectivity, and reproducibility of low abundance protein quantification in both single shot and fractionation. Our results showed that among the tested strategies, MaxQuant in MaxLFQ mode outperformed other strategies in terms of accuracy and precision in both whole proteome and low abundance proteome quantification, whereas the Proteome Discoverer (PD) strategy using SEQUEST as a search engine performed better in terms of quantifiable low abundance proteome coverage. We subsequently applied the PD and MaxLFQ strategies in a blood proteomic dataset and found that many FDA-approved tumor prognostic biomarkers could be identified as well as quantified using the PD strategy, indicating the potential advantage of PD in label-free quantification studies. These results provide a reference for method choice in label-free quantification data analysis. SIGNIFICANCE: Mass spectrometry-based label-free quantification methods play an important role in label-free proteome data analysis. In this study, we evaluated 7 commonly-used label-free quantification methods with respect to the following aspects: missing values, precision, accuracy, selectivity, and reproducibility for low abundance protein quantification. The results showed that, among the strategies evaluated, the PD strategy with SEQUEST as a search engine performed better in terms of low abundance protein coverage. This study provides a reference for method choice in label-free quantification data analysis.

2.
Rapid Commun Mass Spectrom ; 34(2): e8573, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31484223

RESUMO

RATIONALE: Lys-N, also known as lysine-specific metalloendopeptidase, functions as the "sister" enzyme of lysyl endopeptidase (Lys-C) in proteomic research. Its digestion specificity at the N-terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry (MS)-based de novo sequencing of proteins. METHODS: Here we present a complete production process of highly purified Lys-N from dry fruit of Grifola frondosa (maitake mushroom). The purification process includes one step of microfiltration plus one step of UF/DF (ultrafiltration used in tandem with a diafiltration method) recovery and four steps of chromatographic purification. RESULTS: The overall yield of the process was approximately 6.7 mg Lys-N protein/kg dry fruit of G. frondosa. The assay data demonstrated that the purified Lys-N exhibited high enzymatic activity and specificity. CONCLUSIONS: The novel production process provides for the first time the extraction of Lys-N from dry fruit of G. frondosa. The process is also stable and scalable, and provides an economic way of producing the enzyme in large quantities for MS-based proteomics and other biological studies.

3.
J Proteomics ; 210: 103545, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31626998

RESUMO

Targeting specific ubiquitin E3 ligase for degradation of disease-driven protein has recently been an important concept for cancer therapy, as exemplified by the case of thalidomide for the treatment of multiple myeloma. E7070, an aryl sulfonamide drug, exhibited anticancer activity by targeting the E3 ligase receptor DCAF15, with RBM39 as the only known substrate. Exploration of additional substrates of E7070 would facilitate elucidation of its mechanism of actions. To this end, we used a strategy combing pSILAC method with two complementary digestion approaches (LysC-Trypsin and LysN-LysArgiNase) to accurately monitor the protein turnover and increase the depth of proteome profiling. Systematically, we showed that E7070 treatment changed turnover rates of 868 proteins (1.5 fold change and p-value <.05). Several proteins displayed accelerated turnover indicating they were potential new substrates of E7070, among which, pre-mRNA splicing factor 39 (PRPF39) had been reported to be overexpressed in certain cancers. We further demonstrated that PRPF39 was ubiquitinated and degraded by E7070 in a DCAF15-dependent manner, and represented a new bona fide substrate of E7070. The degradation of PRPF39 might also be contributed to the anticancer activity of E7070. SIGNIFICANCE: Identification of degraded substrates is difficult because protein abundance is a comprehensive result regulated by protein production and degradation at the same time. Pulsed SILAC (pSILAC), a method to measure protein turnover, would provide higher sensitivity than total protein quantification. In addition, some peptide sequences are not amenable to MS analysis after LysC-Trypsin digestion. LysN-LysargiNase, as a mirror protease combination of LysC-Trypsin, can be complementary for peptide identification with LysC-Trypsin. By combining pSILAC with two complementary digestion approaches (LysC-Trypsin and LysN-LysArgiNase), we systematically investigated E7070-dependent protein degradation. As a result, we found several potential degradation substrates of E7070 including PRPF39. Further, by exploiting a series of biological assays, we demonstrated that E7070 can lead to the ubiquitination and proteasomal degradation of PRPF39 by promoting the recruitment of PRPF39 to the CUL4-DCAF15 E3 ubiquitin ligase.

4.
J Mass Spectrom ; 55(1): e4441, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31840882

RESUMO

Column heating strategy is often applied in nano-high-performance liquid chromatography-mass spectrometer (nanoHPLC-MS) platform for enhancing the analytical efficiency of peptides or proteins. Nonetheless, the influence effects of column heating in peptides or proteins identification still lack of deep understanding. In this study, a systematic comparison of room temperature (RT) and column heating of nanoHPLC was done. Based on the data, under column heating condition, the backpressure of nanoHPLC can be decreased. Due to the increase of resolution, the peak widths of precursor ion were narrowed. As a result, in MS/MS data acquisition part, more time was spared for MS1 detecting and MS2 fragmenting, which eventually resulted in increased identification of peptides and proteins. Moreover, we also proposed the application scope of column heating by evaluating its influence on sample detection. On one hand, column heating significantly increased the identification of membrane proteins due to more efficient elution of highly hydrophobic peptides compared with RT. On the other hand, heating was not suitable for analyzing short or/and hydrophilic peptides with low retention time, which would be eluted out during sample loading process under high temperature and missed by mass spectrometric detection. In conclusion, our study provides a reference for rational application of column heating in proteomics research.

5.
J Proteomics ; 213: 103614, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31846764

RESUMO

Lysine methylation is a widespread protein post-translational modification showing essentialities in versatile cellular process. EZH2, a methyltransferase specifically trimethylates the lysine 27 of histone H3 and its aberrance in several cancers promotes the development of its inhibitors against hematological tumors. In this study, we presented a deep exploration of lysine mono-, di- and trimethylomes in EZH2 wild-type and Y641 mutant lymphoma cell lines. Our results showed that several substrates were modified in different methylation levels. Moreover, these methylated lysine residues could also undergo other types of PTMs. Combined with the differences proved in protein expression, lysine acetylation, lysine ubiquitylation and protein N-termianl acetylation level, our study underlined the substrate specificity of lysine methylation and its crosstalk with other types of PTMs. Totally, our study raised new insights into the global cellular methylation features in hematological cell lines, which provided further inspects into the distribution and function of lysine methylation. SIGNIFICANCE: Our study showed the global landscape of mono-, di- and trimethylomes in the EZH2-aberrant DLBCL cell lines, revealing the molecular characteristics of lysine methylation. Combined with the protein abundance and potential crosstalk among different types of PTMs, our study raised new insights into the global cellular methylation features in hematological tumors and provided further inspects into the distribution and function of lysine methylation.

6.
Anal Chem ; 91(22): 14522-14529, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31634432

RESUMO

Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies have been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into poly(allylamine)-based C-terminomics (termed as LAACTer). In this strategy, we leveraged LysargiNase, a protease with cleavage specificity N-terminal to Lys and Arg residues, to cover previously unidentifiable C-terminome and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using the original workflow. Compared with the largest human C-terminome data sets (containing 800-900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. In a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative study for identification of GluC-cleaved products, LAACTer quantified 300% more C-terminal peptides than the original workflow. Using LAACTer and the original workflow, we performed global analysis for the C-terminal sequences of 293T cell. The original and processed C-termini displayed distinct sequence patterns, implying the "C-end rules" that regulates protein stability could be more complex than just amino acid motifs. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini.

7.
Mol Cell Proteomics ; 18(2): 391-405, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30420486

RESUMO

The open (mass tolerant) search of tandem mass spectra of peptides shows great potential in the comprehensive detection of post-translational modifications (PTMs) in shotgun proteomics. However, this search strategy has not been widely used by the community, and one bottleneck of it is the lack of appropriate algorithms for automated and reliable post-processing of the coarse and error-prone search results. Here we present PTMiner, a software tool for confident filtering and localization of modifications (mass shifts) detected in an open search. After mass-shift-grouped false discovery rate (FDR) control of peptide-spectrum matches (PSMs), PTMiner uses an empirical Bayesian method to localize modifications through iterative learning of the prior probabilities of each type of modification occurring on different amino acids. The performance of PTMiner was evaluated on three data sets, including simulated data, chemically synthesized peptide library data and modified-peptide spiked-in proteome data. The results showed that PTMiner can effectively control the PSM FDR and accurately localize the modification sites. At 1% real false localization rate (FLR), PTMiner localized 93%, 84 and 83% of the modification sites in the three data sets, respectively, far higher than two open search engines we used and an extended version of the Ascore localization algorithm. We then used PTMiner to analyze a draft map of human proteome containing 25 million spectra from 30 tissues, and confidently identified over 1.7 million modified PSMs at 1% FDR and 1% FLR, which provided a system-wide view of both known and unknown PTMs in the human proteome.


Assuntos
Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Bases de Dados de Proteínas , Humanos , Ferramenta de Busca , Software
8.
Nat Commun ; 9(1): 4770, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425250

RESUMO

Ubiquitin-specific protease 14 (USP14) is one of the major proteasome-associated deubiquitinating enzymes critical for proteome homeostasis. However, substrates of USP14 remain largely unknown, hindering the understanding of its functional roles. Here we conduct a comprehensive proteome, ubiquitinome and interactome analysis for USP14 substrate screening. Bioinformatics analysis reveals broad new potential roles of USP14, especially in lipid and carbohydrate metabolism. Among the potential substrates identified, we show that fatty acid synthase (FASN), a key enzyme involved in hepatic lipogenesis, is a bona fide substrate of USP14. USP14 directly interacts with and increases FASN stability. As a result, overexpression of USP14 promotes liver triglyceride accumulation in C57BL/6 mice, whereas genetic ablation or pharmacological inhibition of USP14 ameliorates hepatosteatosis, hyperglycemia and insulin resistance in obese mice. In conclusion, our findings reveal for the first time an indispensable role of USP14 in hepatosteatosis through FASN stabilization.


Assuntos
Ácido Graxo Sintases/metabolismo , Proteoma , Ubiquitina Tiolesterase/metabolismo , Animais , Metabolismo dos Carboidratos , Biologia Computacional , Técnicas de Silenciamento de Genes , Homeostase , Humanos , Hiperglicemia , Resistência à Insulina , Lipogênese , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Triglicerídeos/análise , Ubiquitina Tiolesterase/genética , Regulação para Cima
10.
Cell Chem Biol ; 25(8): 984-995.e6, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-29887264

RESUMO

Coenzyme A (CoA) esters of short fatty acids (acyl-CoAs) function as key precursors for the biosynthesis of various natural products and the dominant donors for lysine acylation. Herein, we investigated the functional interplay between beneficial and adverse effects of acyl-CoA supplements on the production of acyl-CoA-derived natural products in microorganisms by using erythromycin-biosynthesized Saccharopolyspora erythraea as a model: accumulation of propionyl-CoA benefited erythromycin biosynthesis, but lysine propionylation inhibited the activities of important enzymes involved in biosynthetic pathways of erythromycin. The results showed that the overexpression of NAD+-dependent deacylase could circumvent the inhibitory effects of high acyl-CoA concentrations. In addition, we demonstrated the similar lysine acylation mechanism in other acyl-CoA-derived natural product biosynthesis, such as malonyl-CoA-derived alkaloid and butyryl-CoA-derived bioalcohol. These observations systematically uncovered the important role of protein acylation on interaction between the accumulation of high concentrations of acyl-CoAs and the efficiency of their use in metabolic pathways.


Assuntos
Acil Coenzima A/metabolismo , Produtos Biológicos/metabolismo , Vias Biossintéticas , Eritromicina/metabolismo , Saccharopolyspora/enzimologia , Saccharopolyspora/metabolismo , Acilação , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Saccharopolyspora/química , Metabolismo Secundário
11.
Cell Res ; 28(6): 625-643, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29808012

RESUMO

Cellular senescence is a fundamental cell fate playing a significant role throughout the natural aging process. However, the molecular determinants distinguishing senescence from other cell-cycle arrest states such as quiescence and post-mitotic state, and the specified mechanisms underlying cell-fate decisions towards senescence versus cell death in response to cellular stress stimuli remain less understood. Employing multi-omics approaches, we revealed that switching off the specific mitochondrial processing machinery involving the peptidase IMMP2L serves as the foundation of the senescence program, which was also observed during the mammalian aging process. Mechanistically, we demonstrate that IMMP2L processes and thus activates at least two substrates, mitochondrial metabolic enzyme glycerol-3-phosphate dehydrogenase (GPD2) and cell death regulator apoptosis-inducing factor (AIF). For cells destined to senesce, concerted shutdown of the IMMP2L-GPD2 and IMMP2L-AIF signaling axes collaboratively drives the senescent process by reprogramming mitochondria-associated redox status, phospholipid metabolism and signaling network, and simultaneously blocking cell death under oxidative stress conditions.


Assuntos
Fator de Indução de Apoptose/metabolismo , Senescência Celular , Endopeptidases/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Transdução de Sinais , Envelhecimento , Animais , Morte Celular , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Estresse Oxidativo
12.
Cancer Lett ; 418: 97-108, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29331417

RESUMO

Receptor interacting protein kinase 3 (RIP3) is a critical regulator of programmed necrotic cell death. Here, we observed that RIP3 was significantly down-regulated in esophageal cancer. And its remaining expression was associated with better response to chemotherapy and prolonged survival. Notably, re-expression of kinase-dead RIP3 also restored cisplatin sensitivity, suggesting that some roles of RIP3 beyond necroptosis may be involved in cisplatin-based chemosensitivity. To investigate the mechanisms, a large-scale quantitative proteomics study was performed after cisplatin treatment in RIP3-knockdown cells. In total, approximately 7000 protein groups were confidently identified, with a false discovery rate of 0.21% at the protein level. Of these proteins, 685 displayed RIP3-dependent changes in abundance. Bioinformatics analyses indicated that DNA repair pathway was stimulated after RIP3 depletion. Functional studies showed that deficient RIP3 upregulated FOSL1 and POLD1 through activation of the HSP90/CDC37 complex and ERK phosphorylation in multiple cell lines. Furthermore, via inhibition of the HSP90/CDC37 complex, ERK and FOSL1 reversed the cisplatin resistance phenotype. These results suggest that RIP3 regulates cisplatin sensitivity through both pronecrotic and non-necrotic functions. RIP3 may be a potential marker for predicting chemosensitivity.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Proteínas de Choque Térmico HSP90/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Idoso , Animais , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA/genética , Regulação para Baixo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Feminino , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteômica/métodos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
13.
J Proteome Res ; 16(9): 3460-3469, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28730820

RESUMO

Lysine methylation plays important roles in structural and functional regulation of chromatin. Although trypsin is the most widely used protease in mass spectrometry-based proteomic analysis for lysine methylation substrates, the proteolytic activity of trypsin on dimethylated lysine residues remains an arguable issue. In this study, we tested the ability of trypsin to cleave dimethylated lysine residues in synthetic peptides, purified albumin, and whole cell lysate, and found that the C-terminal of dimethylated lysine residue could be cleaved in a protein sequence-dependent manner. Kinetic studies revealed that the optimal digestion time and enzyme-to-substrate ratio for the cleavage of dimethylated lysine by trypsin was around 16 h and 1:50, respectively. We further showed the tryptic C-terminal lysine-dimethylated (C-Kme2) peptides could contribute to a significant portion of substrate identification in the proteomic study, which utilizes the chemical dimethylation labeling approach. More than 120 tryptic C-Kme2 peptides (7% of total peptides identified) were identified in chemically lysine-dimethyl-labeled HeLa whole cell lysate by a single-shot nanoflow high performance liquid chromatography with tandem mass spectrometry (nano-HPLC-MS/MS) analysis. Moreover, in an assay for substrate identification of protease Glu-C using stable isotope dimethyl labeling approach, our data showed the tryptic C-Kme2 peptides accounted for more than 13% of total tryptic peptides. Additionally, our in vivo methylome profiling data revealed some C-Kme2 peptides, which is of importance to identification and quantification of biologically relevant protein and lysine-methylated site. Therefore, we reason that the tryptic peptides bearing C-terminal dimethylated lysine need to be considered in the mass spectrometric analysis of lysine dimethylation.


Assuntos
Lisina/metabolismo , Fragmentos de Peptídeos/análise , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Marcação por Isótopo/métodos , Cinética , Lisina/química , Metilação , Proteólise , Serina Endopeptidases/química , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Espectrometria de Massas em Tandem , Tripsina/química
14.
Artigo em Inglês | MEDLINE | ID: mdl-28368805

RESUMO

IEF LC-MS/MS is an analytical method that incorporates a two-step sample separation prior to MS identification of proteins. When analyzing complex samples this preparatory separation allows for higher analytical depth and improved quantification accuracy of proteins. However, cost and analysis time are greatly increased as each analyzed IEF fraction is separately profiled using LC-MS/MS. We propose an approach that selects a subset of IEF fractions for LC-MS/MS analysis that is highly informative in the context of a group of proteins of interest. Specifically, our method allows a significant reduction in cost and instrument time as compared to the standard protocol of running all fractions, with little compromise to coverage. We develop algorithmics to optimize the selection of the IEF fractions on which to run LC-MS/MS. We translate the fraction optimization task to Minimum Set Cover, a well-studied NP-hard problem. We develop heuristic solutions and compare them in terms of effectiveness and running times. We provide examples to demonstrate advantages and limitations of each algorithmic approach. Finally, we test our methodology by applying it to experimental data obtained from IEF LC-MS/MS analysis of yeast and human samples. We demonstrate the benefit of this approach for analyzing complex samples with a focus on different protein sets of interest.


Assuntos
Cromatografia Líquida , Focalização Isoelétrica , Proteômica , Espectrometria de Massas em Tandem , Linhagem Celular Tumoral , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Análise por Conglomerados , Custos e Análise de Custo , Humanos , Focalização Isoelétrica/economia , Focalização Isoelétrica/métodos , Proteômica/economia , Proteômica/métodos , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos
15.
Sci Rep ; 7: 41089, 2017 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-28112229

RESUMO

Chronic hepatitis B virus (HBV) infection is partly responsible for hepatitis, fatty liver disease and hepatocellular carcinoma (HCC). HBV core protein (HBc), encoded by the HBV genome, may play a significant role in HBV life cycle. However, the function of HBc in the occurrence and development of liver disease is still unclear. To investigate the underlying mechanisms, HBc-transfected HCC cells were characterized by multi-omics analyses. Combining proteomics and metabolomics analyses, our results showed that HBc promoted the expression of metabolic enzymes and the secretion of metabolites in HCC cells. In addition, glycolysis and amino acid metabolism were significantly up-regulated by HBc. Moreover, Max-like protein X (MLX) might be recruited and enriched by HBc in the nucleus to regulate glycolysis pathways. This study provides further insights into the function of HBc in the molecular pathogenesis of HBV-induced diseases and indicates that metabolic reprogramming appears to be a hallmark of HBc transfection.


Assuntos
Carcinoma Hepatocelular/genética , Vírus da Hepatite B/genética , Neoplasias Hepáticas/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Vírus da Hepatite B/patogenicidade , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Metabolômica , Proteômica , Proteínas do Core Viral/genética
16.
J Proteome Res ; 15(12): 4696-4708, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27804304

RESUMO

Propionylation at protein lysine residue is characterized to be present in both eukaryotic and prokaryotic species. However, the majority of lysine propionylation substrates still remain largely unknown. Using affinity enrichment and mass-spectrometric-based proteomics, we identified 1467 lysine propionylation sites in 603 proteins in E. coli. Quantitative propionylome analysis further revealed that global lysine propionylation level was drastically increased in response to propionate treatment, a carbon source for many microorganisms and also a common food preservative. The results indicated that propionylation may play a regulatory role in propionate metabolism and propionyl-CoA degradation. In contrast with lysine acetylation and succinylation, our results revealed that the lysine propionylation level of substrates showed an obvious decrease in response to high glucose, suggesting a distinct role of propionylation in bacteria carbohydrate metabolism. This study further showed that bacterial lysine deacetylase CobB and acetyltransferase PatZ could also have regulatory activities for lysine propionylation in E. coli. Our quantitative propionylation substrate analysis between cobB wild-type and cobB knockout strain led to the identification of 13 CobB potentially regulated propionylation sites. Together, these findings revealed the broad propionylation substrates in E. coli and suggested new roles of lysine propionylation in bacterial physiology.


Assuntos
Escherichia coli/metabolismo , Lisina/metabolismo , Propionatos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Acetiltransferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Sirtuínas/fisiologia
17.
Sci Rep ; 6: 31795, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27561356

RESUMO

To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during in vitro maturation.


Assuntos
Oócitos/metabolismo , Proteômica/métodos , Animais , Búfalos , Catálise , Bovinos , Técnicas de Cultura de Células , Biologia Computacional , Células do Cúmulo/metabolismo , Feminino , Perfilação da Expressão Gênica , Espectrometria de Massas , Metáfase , Microtúbulos/metabolismo , Oogênese , Fosforilação Oxidativa , Peptídeos , Proteoma , Ribossomos/metabolismo , Tripsina/química
18.
J Proteome Res ; 15(6): 2060-71, 2016 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27183143

RESUMO

Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria.


Assuntos
Metabolismo Energético , Escherichia coli/metabolismo , Lisina/metabolismo , Malonatos/metabolismo , Proteoma/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ciclo do Ácido Cítrico , Biologia Computacional , Ácidos Graxos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos
19.
PLoS One ; 10(2): e0115862, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25723528

RESUMO

The human serum proteome has been extensively screened for biomarkers. However, the large dynamic range of protein concentrations in serum and the presence of highly abundant and large molecular weight proteins, make identification and detection changes in the amount of low-molecular weight proteins (LMW, molecular weight ≤ 30kDa) difficult. Here, we developed a gel-filter method including four layers of different concentration of tricine SDS-PAGE-based gels to block high-molecular weight proteins and enrich LMW proteins. By utilizing this method, we identified 1,576 proteins (n = 2) from 10 µL serum. Among them, 559 (n = 2) proteins belonged to LMW proteins. Furthermore, this gel-filter method could identify 67.4% and 39.8% more LMW proteins than that in representative methods of glycine SDS-PAGE and optimized-DS, respectively. By utilizing SILAC-AQUA approach with labeled recombinant protein as internal standard, the recovery rate for GST spiked in serum during the treatment of gel-filter, optimized-DS, and ProteoMiner was 33.1 ± 0.01%, 18.7 ± 0.01% and 9.6 ± 0.03%, respectively. These results demonstrate that the gel-filter method offers a rapid, highly reproducible and efficient approach for screening biomarkers from serum through proteomic analyses.


Assuntos
Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Espectrometria de Massas , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma , Proteômica/métodos
20.
Dev Cell ; 32(1): 68-81, 2015 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-25556658

RESUMO

The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.


Assuntos
Autoantígenos/metabolismo , Ciclo Celular/fisiologia , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Fosfatase 1/metabolismo , Serina/metabolismo , Western Blotting , Proteína Quinase CDC2 , Proteína Centromérica A , Cromatina/genética , Imunofluorescência , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Mitose/fisiologia , Nucleossomos , Fosforilação
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