细胞培养条件下稳定同位素标记技术(stable isotope labeling by amino acids in cell culture,SILAC)是一种简单的体内标记策略,是基于质谱的定量蛋白质组学技术中的一个有力工具。SILAC技术不会引起蛋白质互作研究中的假阳性结果,使...细胞培养条件下稳定同位素标记技术(stable isotope labeling by amino acids in cell culture,SILAC)是一种简单的体内标记策略,是基于质谱的定量蛋白质组学技术中的一个有力工具。SILAC技术不会引起蛋白质互作研究中的假阳性结果,使细胞信号转导动态变化研究成为可能,并且能够直接测量蛋白质含量。SILAC技术已经应用于一系列的生物学研究中,近来已被成功应用到整体动物水平的定量蛋白质组研究。本文综述了SILAC技术的基本原理、优点和应用进展。展开更多
Most type 2 diabetics are accompanied with deficiency of insulin secretion and hyperglycemia. It has reported that glucose-stimulated insulin secretion of β cell (GSIS)
17-β-estradiol (estrogen) is a steroid hormone important to human development;however, high levels of this molecule are associated with increased risk of breast cancer primarily due to estrogen’s ability to bind and...17-β-estradiol (estrogen) is a steroid hormone important to human development;however, high levels of this molecule are associated with increased risk of breast cancer primarily due to estrogen’s ability to bind and activate the estrogen receptor (ER) and initiate gene transcription. Currently, estrogen mechanisms of action are classified as genomic and non-genomic and occur in an ER-dependent and ER-independent manner. In this study, we examine estrogen signaling pathways, by measuring changes in protein expression as a function of time of exposure to estrogen in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. Using a robust experimental design utilizing isotopic labeling, two-dimensional LC-MS, and bioinformatics analysis, we report genomic and non-genomic ER regulated estrogen responsive proteins. We find a little over 200 proteins differentially expressed after estrogen treatment. Cell proliferation, transcription, actin filament capping and cell to cell signaling are significantly enriched in the MCF-7 cell line alone. Translational elongation and proteolysis are enriched in both cell lines. Subsets of the proteins presented in this study are for the first time directly associated with estrogen signaling in mammary carcinoma cells. We find that estrogen affected the expression of proteins involved in numerous processes that are related to tumorigenesis such as increased cellular division and invasion in an ER-dependent manner. Moreover, we identified negative regulation of apoptosis as a non-genomic process of estrogen. This study complements gene expression studies and highlights the need for both genomic and proteomic analyses in unraveling the complex mechanisms by which estrogen affects progression of breast cancer.展开更多
Brain-derived neurotrophic factor(BDNF)plays an important role in neurodevelopment,synaptic plasticity,learning and memory,and in preventing neurodegeneration.Despite decades of investigations into downstream signalin...Brain-derived neurotrophic factor(BDNF)plays an important role in neurodevelopment,synaptic plasticity,learning and memory,and in preventing neurodegeneration.Despite decades of investigations into downstream signaling cascades and changes in cellular processes,the mechanisms of how BDNF reshapes circuits in vivo remain unclear.This informational gap partly arises from the fact that the bulk of studies into the molecular actions of BDNF have been performed in dissociated neuronal cultures,while the majority of studies on synaptic plasticity,learning and memory were performed in acute brain slices or in vivo.A recent study by Bowling-Bhattacharya et al.,measured the proteomic changes in acute adult hippocampal slices following treatment and reported changes in proteins of neuronal and non-neuronal origin that may in concert modulate synaptic release and secretion in the slice.In this paper,we place these findings into the context of existing literature and discuss how they impact our understanding of how BDNF can reshape the brain.展开更多
The tumor necrosis factor （TNF）-α/NF-kB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The com...The tumor necrosis factor （TNF）-α/NF-kB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The complexity of the TNF-α/NF-kB signaling is in part due to the dynamic protein behaviors of key players in this pathway. In this present work, a dynamic and global view of the signaling components in the nucleus at the early stages of TNF-a/ NF-KB signaling was obtained in HEK293 cells, by a combination of subcellular fractionation and stable isotope la- beling by amino acids in cell culture （SILAC）. The dynamic profile patterns of 547 TNF-α-induced nuclei-associated proteins were quantified in our studies. The functional characters of all the profiles were further analyzed using that Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway annotation. Additionally, many previously unknown effectors of TNF-α/NF-kB signaling were identified, quantified and clustered into differential activation profiles. In- terestingly, levels of Fanconi anemia group D2 protein （FANCD2）, one of the Fanconi anemia family proteins, was found to be increased in the nucleus by SILAC quantitation upon TNF-α stimulation, which was further verified by western blotting and immunofluorescence analysis. This indicates that FANCD2 might be involved in TNF-α/NF-kB signaling through its accumulation in the nucleus. In summary, the combination of subcellular proteomics with quan- titative analysis not only allowed for a dissection of the nuclear TNF-α/NF-kB-signaling pathway, but also provided a systematic strategy for monitoring temporal and spatial changes in cell signaling.展开更多
Cyclin-dependent kinases(CDKs) have become potential targets for treating various diseases, especially cancer. Compound i CDK9 is an excellent and selective CDK9 inhibitor, but its major limitation is the potential to...Cyclin-dependent kinases(CDKs) have become potential targets for treating various diseases, especially cancer. Compound i CDK9 is an excellent and selective CDK9 inhibitor, but its major limitation is the potential toxicity and poor understanding of the underlying mechanism. The PROTAC(proteolysis targeting chimera) degraders of bioactive molecules can significantly induce in vitro and in vivo degradation of their target protein with high selectivity and effectively reduce the dose-limiting toxicity of small molecule drugs. Therefore, we designed and synthesized the bifunctional PROTAC molecules of i CDK9, being used for identifying its previously unknown target and revealing the underlying pharmacological mechanism.The PROTAC bifunctional molecule CD-5 could selectively and significantly degrade CDK9 with low cell toxicity. Therefore, we selected CD-5 as a chemical prober in the SILAC quantitative proteomic analysis, which disclosed that CD-5 could enormously lessen the lysine acetyltransferase KAT6A. Furthermore,KAT6A degradation induced by CD-5 repressed the levels of H3K14Ac and H3K23Ac. Lastly, the streptavidin immunoprecipitation(IP) assay confirmed a direct interaction between KAT6A and i CDK9. Collectively, our results uncover that KAT6A is a potential non-kinase target of i CDK9. Notably, this study also demonstrates that the PROTAC-SILAC strategy is an alternative approach for cellular target identification of bioactive molecules.展开更多
文摘细胞培养条件下稳定同位素标记技术(stable isotope labeling by amino acids in cell culture,SILAC)是一种简单的体内标记策略,是基于质谱的定量蛋白质组学技术中的一个有力工具。SILAC技术不会引起蛋白质互作研究中的假阳性结果,使细胞信号转导动态变化研究成为可能,并且能够直接测量蛋白质含量。SILAC技术已经应用于一系列的生物学研究中,近来已被成功应用到整体动物水平的定量蛋白质组研究。本文综述了SILAC技术的基本原理、优点和应用进展。
文摘Most type 2 diabetics are accompanied with deficiency of insulin secretion and hyperglycemia. It has reported that glucose-stimulated insulin secretion of β cell (GSIS)
文摘17-β-estradiol (estrogen) is a steroid hormone important to human development;however, high levels of this molecule are associated with increased risk of breast cancer primarily due to estrogen’s ability to bind and activate the estrogen receptor (ER) and initiate gene transcription. Currently, estrogen mechanisms of action are classified as genomic and non-genomic and occur in an ER-dependent and ER-independent manner. In this study, we examine estrogen signaling pathways, by measuring changes in protein expression as a function of time of exposure to estrogen in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. Using a robust experimental design utilizing isotopic labeling, two-dimensional LC-MS, and bioinformatics analysis, we report genomic and non-genomic ER regulated estrogen responsive proteins. We find a little over 200 proteins differentially expressed after estrogen treatment. Cell proliferation, transcription, actin filament capping and cell to cell signaling are significantly enriched in the MCF-7 cell line alone. Translational elongation and proteolysis are enriched in both cell lines. Subsets of the proteins presented in this study are for the first time directly associated with estrogen signaling in mammary carcinoma cells. We find that estrogen affected the expression of proteins involved in numerous processes that are related to tumorigenesis such as increased cellular division and invasion in an ER-dependent manner. Moreover, we identified negative regulation of apoptosis as a non-genomic process of estrogen. This study complements gene expression studies and highlights the need for both genomic and proteomic analyses in unraveling the complex mechanisms by which estrogen affects progression of breast cancer.
文摘多药耐药性(multidrug resistance,MDR)是临床肿瘤化疗失败的主要原因之一,定量分析与鉴定食管鳞癌顺铂(cis-diamminedichloroplatinum,CDDP)耐药相关蛋白对阐明食管癌耐药的分子机制具有重要理论意义。本研究采用浓度递增法建立食管癌CDDP耐药细胞系EC9706/CDDP,细胞培养稳定同位素标记(stable isotope labeling with amino acids in cell culture,SILAC)和高效液相-电喷雾串联质谱检测、生物信息学定量分析并鉴定EC9706/CDDP及其母细胞系EC9706的差异蛋白表达谱。EC9706/CDDP细胞呈贴壁性生长但增殖缓慢,多形性、异形性明显,耐药指数为3.23。74种差异表达的蛋白质分子主要包括细胞骨架相关蛋白(20%)、能量代谢相关蛋白(11%)、转录调控及DNA修复相关蛋白(11%)、氧化还原内稳态维持类蛋白(9.5%)、蛋白合成及参与mRNA处理类蛋白(12%)、核糖体结构类蛋白(8.1%)、分子伴侣蛋白(8.1%)、免疫/炎症反应相关蛋白(5.4%)、细胞内转运功能相关蛋白(5.4%)、核组装相关蛋白(2.7%)等。表明食管癌顺铂耐药的发生是多分子参与、多代谢通路失调的复杂过程,差异表达的蛋白分子将有助于深入理解MDR发生的分子机制,并为MDR表型逆转的新型药物设计提供有价值的分子靶点。
基金supported by NIH grants NS034007 and NS047384supported by NIH grants NS21072,and HD23315supported by funds from the Department of Biotechnology,Government of India and the Shanta Wadhwani Foundation
文摘Brain-derived neurotrophic factor(BDNF)plays an important role in neurodevelopment,synaptic plasticity,learning and memory,and in preventing neurodegeneration.Despite decades of investigations into downstream signaling cascades and changes in cellular processes,the mechanisms of how BDNF reshapes circuits in vivo remain unclear.This informational gap partly arises from the fact that the bulk of studies into the molecular actions of BDNF have been performed in dissociated neuronal cultures,while the majority of studies on synaptic plasticity,learning and memory were performed in acute brain slices or in vivo.A recent study by Bowling-Bhattacharya et al.,measured the proteomic changes in acute adult hippocampal slices following treatment and reported changes in proteins of neuronal and non-neuronal origin that may in concert modulate synaptic release and secretion in the slice.In this paper,we place these findings into the context of existing literature and discuss how they impact our understanding of how BDNF can reshape the brain.
基金Abbreviations： TNF （tumor necrosis factor） SILAC （stable isotope label- ing by amino acids in cell culture）+4 种基金 LC-MS/MS （liquid chromatography tandem mass spectrometry） LTQ-Orbitrap （linear ion trap/Orbitrap） HPRD （Human Protein Reference Database） SMARC （SWI/SNF-related matrix-associated actin-dependent regulator chromatin） KEGG （Kyoto Encyclopedia of Genes and Genomes）Acknowledgment We would like to thank Dr/an Scott （Department of Molecular Genetics, University of Toronto）, Dr Chengjian Tu （Vanderbilt Medical Center, Vanderbilt University） and Ya-Wen Tian （Key Laboratory of Systems Biology, Shanghai Institutes for Biologi- cal Sciences） for critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China （30425021, 30521005）, Basic Research Foundation （2006CB910700）, CAS Project （KSCX2-YW-R-106, KSCX1- YW-02） and High-technology Project （2007AA02Z334）.
文摘The tumor necrosis factor （TNF）-α/NF-kB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The complexity of the TNF-α/NF-kB signaling is in part due to the dynamic protein behaviors of key players in this pathway. In this present work, a dynamic and global view of the signaling components in the nucleus at the early stages of TNF-a/ NF-KB signaling was obtained in HEK293 cells, by a combination of subcellular fractionation and stable isotope la- beling by amino acids in cell culture （SILAC）. The dynamic profile patterns of 547 TNF-α-induced nuclei-associated proteins were quantified in our studies. The functional characters of all the profiles were further analyzed using that Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway annotation. Additionally, many previously unknown effectors of TNF-α/NF-kB signaling were identified, quantified and clustered into differential activation profiles. In- terestingly, levels of Fanconi anemia group D2 protein （FANCD2）, one of the Fanconi anemia family proteins, was found to be increased in the nucleus by SILAC quantitation upon TNF-α stimulation, which was further verified by western blotting and immunofluorescence analysis. This indicates that FANCD2 might be involved in TNF-α/NF-kB signaling through its accumulation in the nucleus. In summary, the combination of subcellular proteomics with quan- titative analysis not only allowed for a dissection of the nuclear TNF-α/NF-kB-signaling pathway, but also provided a systematic strategy for monitoring temporal and spatial changes in cell signaling.
基金supported by grants from the National Key R&D Program of China (Nos. 2018YFA0107303 and 2020YFA0908100)the National Natural Science Foundation of China (Nos. 81773600and 82102746)+1 种基金the China Postdoctoral Science Foundation (No.2021M690095)the Fundamental Research Funds for the Central Universities (No. 20720180051)。
文摘Cyclin-dependent kinases(CDKs) have become potential targets for treating various diseases, especially cancer. Compound i CDK9 is an excellent and selective CDK9 inhibitor, but its major limitation is the potential toxicity and poor understanding of the underlying mechanism. The PROTAC(proteolysis targeting chimera) degraders of bioactive molecules can significantly induce in vitro and in vivo degradation of their target protein with high selectivity and effectively reduce the dose-limiting toxicity of small molecule drugs. Therefore, we designed and synthesized the bifunctional PROTAC molecules of i CDK9, being used for identifying its previously unknown target and revealing the underlying pharmacological mechanism.The PROTAC bifunctional molecule CD-5 could selectively and significantly degrade CDK9 with low cell toxicity. Therefore, we selected CD-5 as a chemical prober in the SILAC quantitative proteomic analysis, which disclosed that CD-5 could enormously lessen the lysine acetyltransferase KAT6A. Furthermore,KAT6A degradation induced by CD-5 repressed the levels of H3K14Ac and H3K23Ac. Lastly, the streptavidin immunoprecipitation(IP) assay confirmed a direct interaction between KAT6A and i CDK9. Collectively, our results uncover that KAT6A is a potential non-kinase target of i CDK9. Notably, this study also demonstrates that the PROTAC-SILAC strategy is an alternative approach for cellular target identification of bioactive molecules.
