Protein Microcharacterization
蛋白质微观表征
基本信息
- 批准号:8336708
- 负责人:
- 金额:$ 84.22万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:AKT2 geneATP Synthesis PathwayAcylationAffinityAffinity ChromatographyArachidonic AcidsBindingBiologicalBiological ProcessBiotinylationCD34 geneCategoriesCell AdhesionCell NucleusCellsCleaved cellComplementComplexConsensusConsumptionCore FacilityCoupledCysteineCytoplasmDNA RepairDNA biosynthesisDataDefectDetergentsDevelopmentDominant-Negative MutationEnergy MetabolismEnzymatic BiochemistryExtracellular DomainFailureFatty AcidsFeedbackFlagellaGene ExpressionGenesGerm CellsGlucocorticoid ReceptorGlucoseGlyceraldehyde-3-Phosphate DehydrogenasesGlycolysisGlycoproteinsGuanosine Triphosphate PhosphohydrolasesHair follicle structureHandHistocompatibility TestingHistonesHomeostasisHumanHydroxylamineIn VitroInositolIntramural ResearchIsoenzymesLabelLaboratoriesLactate DehydrogenaseLengthLipidsLocationLondonMAP Kinase GeneMAPK14 geneMaintenanceMale InfertilityMass Spectrum AnalysisMediatingMembraneMembrane MicrodomainsMembrane Protein TrafficMetabolic DiseasesModificationMolecular WeightMusNADHNational Institute of Environmental Health SciencesNuclear Magnetic ResonanceNucleolar ProteinsPalmitatesPathway interactionsPeptidesPhosphorylationPhosphorylation SitePhosphotransferasesPolysaccharidesPost-Translational Modification SitePost-Translational Protein ProcessingPreparationProtein Kinase CProteinsProteolysisProteomicsPublishingPyruvateRegulationReportingResearchResearch PersonnelResidual stateResistanceResourcesRoleSamplingSerineServicesSignal PathwaySignal TransductionSiteSkin NeoplasmsSodiumStem cellsSurface AntigensTechniquesTestingTimeTyrosine Phosphorylation SiteVariantWorkcell motilitycell typecofactorin vivoinhibitor/antagonistmacrophagenucleolinpalmitoylationprotein aminoacid sequenceprotein expressionprotein functionreceptorresearch studysmall hairpin RNAsperm cellsperm functionstreptavidin-agarosethioester
项目摘要
A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Microcharacterization Core Facility (PMCF) with approximately 5000 samples analyzed from.
One large effort is in support of the Protein Expression Core Facility (PECF) and Dr. Bob Petrovich. The Role of the PMCF is to confirm gene expression at the protein level prior to the PECF handing materials over to their users.
Other unpublished projects that are still ongoing include:
Identification of binding partners and sites of post-translational modifications (PTMs) on lipid and inositol kinases - Steve Shears
Identification of posttranslational modification of proteins that may be involved in DNA replication and repair - Sam Wilson and Bob London
Identification of proteins in the BAF complexes under a variety of tissue types and/or conditions - Trevor Archer
Analyses of histone variants - Serena Dudek
Glucocorticoid receptor modifications and binding partners - John Cidlowski
The core also is performing value added research in affinity techniques (Bromodoamin Enrichment)to aid in protein and PTM identifications.
Other published projects or projects with manuscriptis in preparation include:
Identification of RhoA binding partners: Arachidonic acid (AA) stimulates cell adhesion through a p38 MAPK-mediated RhoA signaling pathway. We performed a proteomic screen following AA-treatment identified nucleolin, a multifunctional nucleolar protein, in a complex with the GTPase, RhoA. AA-stimulated cell adhesion was blocked by expression of nucleolin-targeted shRNA. Furthermore, formation of a RhoA/ROCK/p38/nucleolin complex was blocked by expression of dominant negative RhoA. AA-treatment also induced ROCK-dependent serine phosphorylation of nucleolin and translocation of nucleolin from the nucleus to the cytoplasm, where it co-localized with RhoA. These data suggest a new signaling pathway through which the location and post-translational state of nucleolin are modulated.
Phosphorylation of CD34: CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types and has been described as a marker for epidermal stem cells in mouse hair follicles. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309382) following PKC treatment. For this work, we used a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34. -Ken Tomer
Macrophage Palmitoylation: S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine vs. mock treatment conditions. Of 1,183 proteins identified in 4 independent experiments, 80 proteins were significant for S-acylation at false discovery rate(FDR)=0.05, and 101 significant at FDR=0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and 3 presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. -Mike Fessler and Ken Tomer
LDHC: Germ cell-specific lactate dehydrogenase C gene (Ldhc) leads to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC. -Mitch Eddy
Additional projects that have required more than negligible resources include efforts performed with the Armstrong, Birnbaumer, Blackshear, Eling, Freedman, Hall, Hu, and Negishi laboratories.
蛋白质表征方面的各种服务和合作项目已经或正在利用蛋白质微表征核心设施 (PMCF) 进行,并分析了大约 5000 个样品。
其中一项重大工作是支持蛋白质表达核心设施 (PECF) 和 Bob Petrovich 博士。 PMCF 的作用是在 PECF 将材料交给用户之前在蛋白质水平上确认基因表达。
其他仍在进行中的未发布项目包括:
脂质和肌醇激酶上结合伴侣和翻译后修饰 (PTM) 位点的鉴定 - Steve Shears
鉴定可能参与 DNA 复制和修复的蛋白质翻译后修饰 - Sam Wilson 和 Bob London
在各种组织类型和/或条件下鉴定 BAF 复合物中的蛋白质 - Trevor Archer
组蛋白变体分析 - Serena Dudek
糖皮质激素受体修饰和结合伙伴 - John Cidlowski
该核心还在亲和技术(溴多胺富集)方面进行增值研究,以帮助蛋白质和 PTM 鉴定。
其他已发表的项目或正在准备手稿的项目包括:
RhoA 结合伴侣的鉴定:花生四烯酸 (AA) 通过 p38 MAPK 介导的 RhoA 信号通路刺激细胞粘附。我们在 AA 处理后进行了蛋白质组筛选,鉴定出核仁素(一种多功能核仁蛋白)与 GTP 酶 RhoA 的复合物。核仁素靶向 shRNA 的表达可阻断 AA 刺激的细胞粘附。 此外,显性失活 RhoA 的表达阻断了 RhoA/ROCK/p38/核仁素复合物的形成。 AA 处理还诱导核仁素的 ROCK 依赖性丝氨酸磷酸化以及核仁素从细胞核易位到细胞质,在细胞质中与 RhoA 共定位。 这些数据表明了一种新的信号传导途径,通过该途径调节核仁素的位置和翻译后状态。
CD34 的磷酸化:CD34 是一种 I 型跨膜糖蛋白,是一种在多种细胞类型上表达的表面抗原,已被描述为小鼠毛囊中表皮干细胞的标记物。 尽管 CD34 的生物学功能和调节尚不清楚,但人们认为它参与细胞粘附,并可能在信号转导中发挥作用。此外,CD34 被证明对小鼠皮肤肿瘤的发展至关重要,尽管确切的机制仍不清楚。 许多蛋白质的功能和生物活性是通过翻译后修饰来调节的。 CD34 的胞外结构域高度糖基化,但这些聚糖在 CD34 功能中的作用尚不清楚。此外,已报道人 CD34 上有两个酪氨酸磷酸化位点,并且已知 CD34 至少部分被蛋白激酶 C 磷酸化;然而,磷酸化位点的精确位置尚未报道。为了鉴定 CD34 中的特定磷酸化位点并描述蛋白激酶 C 的可能作用,我们在 PKC 处理后鉴定了小鼠 CD34 (aa 309382) 胞内结构域上的磷酸化位点。在这项工作中,我们结合使用了酶促蛋白水解和质谱肽测序。之后测定HEK293F细胞表达的全长小鼠CD34的体内磷酸化位点。然而,观察到的体内磷酸化位点并不是共有的 PKC 位点,但我们的数据表明这些位点之一可能被 AKT2 磷酸化。这些结果表明其他激酶以及 PKC 可能在 CD34 中具有重要的信号传导功能。 ——肯·托默
巨噬细胞棕榈酰化:S-棕榈酰化是特定半胱氨酸残基与脂肪酸棕榈酸酯的可逆翻译后酰化,促进多种生物途径中蛋白质的膜束缚和亚细胞定位。尽管抑制棕榈酰化有望成为操纵蛋白质靶向的一种手段,但由于对棕榈酰化酶学和共有基序的了解有限,该领域的进展受到阻碍。为了确定巨噬细胞中 S-酰化蛋白的补体,我们用羟胺处理 RAW 264.7 巨噬细胞膜以裂解酰基硫酯,然后对新暴露的巯基进行生物素化,并进行链霉亲和素-琼脂糖亲和层析。在通过 LC-MS/MS 鉴定的蛋白质中,通过光谱计数确定 S-酰化状态,以评估羟胺与模拟处理条件下的富集情况。在 4 个独立实验中鉴定出的 1,183 个蛋白质中,80 个蛋白质在错误发现率 (FDR)=0.05 时对 S-酰化具有显着性,101 个在 FDR=0.10 时具有显着性。候选S-酰基蛋白从多个功能类别中被鉴定出来,包括膜运输、信号传导、转运蛋白和受体。其中有 29 种蛋白质先前被生化证实为棕榈酰化,45 种蛋白质先前在蛋白质组学筛选中报告为假定的 S-酰基蛋白,24 种先前与棕榈酰化无关,还有 3 种推测为假阳性。我们之前在耐巨噬细胞去污剂膜中鉴定出近一半的候选者,这表明棕榈酰化促进了巨噬细胞中蛋白质的脂筏定位。 ——迈克·费斯勒和肯·托默
LDHC:生殖细胞特异性乳酸脱氢酶 C 基因 (Ldhc) 由于精子功能缺陷而导致男性不育,包括精子 ATP 水平快速下降、前向活力下降以及无法发展过度活跃的活力。我们假设缺乏 LDHC 会通过反馈抑制来破坏糖酵解,要么导致对 3-磷酸甘油醛脱氢酶、精子 (GAPDHS) 活性至关重要的 NAD(+) 辅因子更新缺陷,要么导致丙酮酸积累。为了测试这些假设,使用核磁共振分析来实时跟踪标记底物的利用。我们发现,在缺乏 LDHC 的精子中,葡萄糖消耗受到干扰,但 NAD:NADH 比率和丙酮酸水平没有变化,并且丙酮酸迅速代谢为乳酸。此外,乳酸脱氢酶(LDH)抑制剂草酸钠治疗引起的代谢紊乱与缺乏LDHC引起的代谢紊乱不同。这支持了我们之前的结论,即 LDHA(一种存在于鞭毛主片中的 LDH 同工酶)是缺乏 LDHC 的精子中残留 LDH 活性的原因,但也表明 LDHC 在维持精子能量代谢方面具有额外的作用。通过免疫共沉淀结合质谱法,我们鉴定了 27 种与 LDHC 相关的蛋白质。这些蛋白质中的大多数与 ATP 合成、利用、运输和/或隔离有关。这使我们推测,LDHC 除了在糖酵解中发挥作用外,还是参与 ATP 稳态的复合物的一部分,而缺乏 LDHC 的精子中该复合物会被破坏。 ——米奇·艾迪
其他需要大量资源的项目包括与 Armstrong、Birnbaumer、Blackshear、Eling、Freedman、Hall、Hu 和 Negishi 实验室合作开展的工作。
项目成果
期刊论文数量(0)
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Jason Williams其他文献
Jason Williams的其他文献
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{{ truncateString('Jason Williams', 18)}}的其他基金
Determining the role of Pcdh10a in zebrafish migratory neural crest cells
确定 Pcdh10a 在斑马鱼迁移神经嵴细胞中的作用
- 批准号:
9269183 - 财政年份:2015
- 资助金额:
$ 84.22万 - 项目类别:
Methods to Compare Mechanisms of Action in Substance Use Programs
比较药物使用计划中作用机制的方法
- 批准号:
8210920 - 财政年份:2011
- 资助金额:
$ 84.22万 - 项目类别:
Methods to Compare Mechanisms of Action in Substance Use Programs
比较药物使用计划中作用机制的方法
- 批准号:
8044943 - 财政年份:2011
- 资助金额:
$ 84.22万 - 项目类别:
The effect of oxidative stress on muscle damage and functional senesence.
氧化应激对肌肉损伤和功能衰老的影响。
- 批准号:
7497989 - 财政年份:2007
- 资助金额:
$ 84.22万 - 项目类别:
The effect of oxidative stress on muscle damage and functional senesence.
氧化应激对肌肉损伤和功能衰老的影响。
- 批准号:
7276332 - 财政年份:2007
- 资助金额:
$ 84.22万 - 项目类别: