PHOSPHORYLATION OF RRN3

RRN3 的磷酸化

• 批准号: 7602042
• 负责人: LAWRENCE I ROTHBLUM
• 金额: $ 0.54万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602042
• 关键词: Alkaline Phosphatase; Amino Acids; Bite; CCL26 gene; Cells; Characteristics; Complex; Computer Retrieval of Information on Scientific Projects Database; Core Assembly; Data; Digestion; Dissociation; Electrons; Endopeptidases; Enzymes; Funding; Genes; Genetic Transcription; Goals; Grant; Institution; Ions; Mass Spectrum Analysis; Measurement; Measures; Methods; Parents; Peptide Hydrolases; Peptides; Personal Satisfaction; Phosphopeptides; Phosphoric Acids; Phosphorylated Peptide; Phosphorylation; Phosphorylation Site; Polymerase; Proteins; Publications; RNA Polymerase I; RNA Polymerase II; Recombinant DNA; Research; Research Personnel; Resolution; Resources; Ribosomal DNA; Ribosomal RNA; Role; Running; SHFM1 gene; Scanning; Signal Transduction; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Testing; Time; Transcriptional Regulation; Trypsin; United States National Institutes of Health; Work; base; chymotrypsin; dalton; glutamyl endopeptidase; imidazole-4-acetic acid; improved; insight; protein aminoacid sequence; protein protein interaction; research study; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Many of the protein-protein interactions involved in transcription have been elucidated from studies on genes transcribed by RNA polymerase II, and serve as paradigms for understanding the various mechanisms involved in the regulation of transcription. However, transcription by RNA polymerase I involves an independently evolved group of transcription factors. Understanding the interactions among these factors may provide new insights into the mechanisms of gene transcription. The long-term goal of our work is to determine the protein-protein interactions that regulate ribosomal RNA (rDNA) transcription. The ability of RNA polymerase I (pol I) itself to participate in rDNA transcription is regulated by the assembly of the core subunits of pol I with different polymerase associated factors. Previous data show at least two polymerase-associated factors are required for transcription. One of these, Rrn3, must be phosphorylated to function in transcription.1,2 We hypothesize that the state of Rrn3 phosphorylation regulates the formation of the Rrn3-RNA polymerase I complex that is required for transcription. The goal of this project is to test this hypothesis by identifying the phosphorylated residues in Rrn3 and determining their role(s) in transcription. Part of this goal includes the characterization of the interactions between Rrn3 and the other components of the rDNA transcription apparatus. Definitive identification of phosphorylated peptides and specific amino acids can be obtained by MALDI-TOF-MS, LC-MS/MS or nanospray-MSn analysis. Peptides generated with trypsin or relatively nonspecific proteases, chymotrypsin and V8 protease, from active Rrn3 purified from exponentially growing cells will be analyzed by mass spectrometry to identify the phosphorylation sites. It may be feasible to facilitate the MS analyses by carrying out 2D-TLC to identify tryptic fragments and then subject just those spots to MS/MS (similar to LC-MS). In preliminary experiments, we have determined that we can purify enough protein by this method to carry out subsequent MS analysis. Furthermore, tryptic peptides from Rrn3 were analyzed at the BUSM MSR by electrospray FTMS using the newly developed ESI-qQq-FTMS. One peptide, tryptic peptide T36-48, was phosphorylated, but the abundance of this peptide in the spectrum was ~3% of the most abundant ion. The region around m/z 404 was isolated using Q1 and accumulated in Q2 prior to electron capture dissociation and collisionally activated dissociation. The combined b/y and c/z cleavages from the two methods multiply confirmed the site of phosphorylation. Overall, the magenerated on the basis of ECD and CAD FTMS analysis is well populated, but further work using IMAC to enrich the phosphopeptides prior to ECD, and using different enzymes such as Asp-N and Glu-C should improve the coverage. The accuracy of mass measurements with delayed extraction and a reflector analyzer (accurate to ~10 parts per million with intense peaks), make it possible to unambiguously assign phosphopeptides of a protein with a known sequence such as Rrn3. Peptides measuring 80 Da higher in mass than their predicted sequence are tentatively marked as phosphopeptides to be confirmed by MS/MS. Confirmation of phosphorylation can also be obtained by comparing the MALDI spectra obtained before and after digestion with alkaline phosphatase. Phosphorylated peptides can also be analyzed using nanoflow-HPLC-MS/MS. When the MS/MS is run in positive ion mode, peptide whole masses (MS) are measured and data for peptide sequencing is obtained by operating a Q-TOF-MS in the data-dependent mode and cycling through MS and MS/MS experiments. In a neutral ion loss experiment a precursor ion scan is used to identify those parent ions that undergo the characteristic 98 Dalton ion neutral loss corresponding to the elimination of phosphoric acid. At this time we have been able to achieve ~58% coverage of Rrn3 isolated from Sf9 cells and have identified four phosphopeptides. Two of these peptides, ALENDFFNSPPR and EGDVDVSDSDDEDDN LPANFDTCHR, are the same as those identified by Schlosser et al. We are now working with Prof. OConnor to achieve complete PTM analysis of Rrn3 using top-down MS/MS, high resolution bottom-up MS/MS, and electron capture dissociation on his FTMS. While Rrn3 is clearly a bit large for most Top-Down experiments, his new ESIi-qQq-FTMS has the ability to selectively accumulate ions and perform a wide variety of MS/MS methods that may improve and simplify this analysis. Already some signal has been observed and some phosphopeptides from Rrn3 have been observed by ESI-FTMS. A publication is being prepared.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 许多参与转录的蛋白质-蛋白质相互作用已经从RNA聚合酶II转录的基因的研究中阐明，并作为理解转录调控中所涉及的各种机制的范例。然而，RNA聚合酶I的转录涉及一组独立进化的转录因子。 了解这些因素之间的相互作用可能会提供新的见解基因转录的机制。 我们工作的长期目标是确定调节核糖体RNA（rDNA）转录的蛋白质-蛋白质相互作用。RNA聚合酶I（pol I）自身参与rDNA转录的能力受pol I的核心亚基与不同聚合酶相关因子的组装调节。先前的数据显示至少两个聚合酶相关因子是转录所必需的。其中之一，Rrn3，必须磷酸化才能在转录中发挥作用。1，2我们假设Rrn3磷酸化状态调节转录所需的Rrn3-RNA聚合酶I复合物的形成。 本项目的目标是通过鉴定Rrn3中的磷酸化残基并确定其在转录中的作用来验证这一假设。这一目标的一部分包括Rrn3和rDNA转录装置的其他组件之间的相互作用的表征。 通过MALDI-TOF-MS、LC-MS/MS或纳米喷雾-MSn分析可以获得磷酸化肽和特定氨基酸的连续鉴定。将通过质谱法分析用胰蛋白酶或相对非特异性蛋白酶、胰凝乳蛋白酶和V8蛋白酶从指数生长细胞纯化的活性Rrn3产生的肽，以鉴定磷酸化位点。 通过进行2D-TLC以鉴别胰蛋白酶片段，然后仅对这些斑点进行MS/MS（类似于LC-MS），可能有助于MS分析。在初步实验中，我们已经确定，我们可以通过这种方法纯化足够的蛋白质进行后续的MS分析。 此外，使用新开发的ESI-qQq-FTMS，通过电喷雾FTMS在BUSM MSR分析了Rrn3的胰蛋白酶肽。 一种肽（胰蛋白酶肽T36 - 48）被磷酸化，但该肽在光谱中的丰度约为最丰富离子的3%。 使用Q1分离m/z 404周围的区域，并在电子捕获解离和碰撞活化解离之前在Q2中累积。 来自两种方法的组合B/y和c/z切割多重确认了磷酸化位点。 总的来说，基于ECD和CAD FTMS分析的magenerated是很好的填充，但进一步的工作使用IMAC富集的磷酸肽之前，ECD，并使用不同的酶，如Asp-N和Glu-C应提高覆盖率。 延迟提取和反射器分析仪的质量测量准确度（精确到约百万分之10，具有强峰），使得可以明确地分配具有已知序列（如Rrn3）的蛋白质的磷酸肽。 将质量比其预测序列高80 Da的肽暂时标记为磷酸肽，以通过MS/MS确认。也可以通过比较碱性磷酸酶消化前后获得的MALDI光谱来确认磷酸化。 也可以使用纳米流-HPLC-MS/MS分析磷酸化肽。当MS/MS以正离子模式运行时，测量肽整体质量（MS），并通过以数据依赖性模式操作Q-TOF-MS并循环通过MS和MS/MS实验来获得肽测序的数据。 在中性离子损失实验中，使用前体离子扫描来识别经历对应于磷酸消除的特征性98道尔顿离子中性损失的那些母离子。在这个时候，我们已经能够实现从Sf9细胞分离的Rrn3的~58%的覆盖率，并确定了四个磷酸肽。这些肽中的两种，ALENDFFNSPPR和EGDVDVSDSDDEDDNLPANFDT3，与Schlosser等人鉴定的肽相同。 我们现在与O教授合作康纳在他的FTMS上使用自上而下的MS/MS、高分辨率自下而上的MS/MS和电子捕获解离来实现Rrn3的完整PTM分析。虽然Rrn3对于大多数自上而下的实验来说显然有点大，但他的新ESIi-qQq-FTMS能够选择性地积累离子，并执行各种各样的MS/MS方法，可以改进和简化这种分析。 已经观察到一些信号，并且已经通过ESI-FTMS观察到来自Rrn3的一些磷酸肽。 正在编写一份出版物。