Protein Microcharacterization

蛋白质微观表征

基本信息

批准号：
9550633
负责人：
Jason Williams
金额：
$ 109.18万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
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：
https://reporter.nih.gov/project-details/9550633
关键词：
Allergens Binding Collaborations Complex Core Facility DNA DNA Repair DNA-protein crosslink Disease Dust Endoribonucleases Enzymes Family Gene Expression Genes Hand Head Hydrolase In Vitro Intramural Research LIG4 gene Laboratories Life Ligase Link London Mass Spectrum Analysis Molecular Weight Moon Motor Neuron Disease National Institute of Environmental Health Sciences Pathway interactions Peptides Pharmaceutical Preparations Phosphorylation Site Phosphotransferases Polynucleotide 5&apos -Hydroxyl-Kinase Post-Translational Modification Site Post-Translational Protein Processing Principal Investigator Process Protein Analysis Protein-Serine-Threonine Kinases Proteins Publications Publishing RNA RNA Processing RNA Splicing Raja Reaction Recombinant Proteins Recruitment Activity Regulation Reporting Research Personnel Research Support Resolution Resources Ribosomal RNA Robin bird Role Saccharomyces cerevisiae Sampling Scientist Services Site Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Support Groups Topoisomerase Transact Work X-linked intellectual disability cooking crosslink experimental study exportin 1 protein genotoxicity insight member multicatalytic endopeptidase complex novel pathogen programs protein expression rRNA Precursor response tyrosyl-DNA phosphodiesterase

项目摘要

A variety of service and collaborative projects in protein characterization have been or are being carried out within the Mass Spectrometry Research and Support Group with approximately 4000 samples analyzed from 39 scientists representing 23 principal investigators or core heads from 6 laboratory branches and the DNTP. 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 Other published and unpublished projects that are still ongoing include: Characterization of dust and proteins from allergens in collaboration - Don Cook, Geoffrey Mueller, and Robert London Characterization of protein:protein cross-links in RNA processing enzymes Robin Stanley Characterization of post-translational modifications on the CRM1 protein Raja Jothi Characterization of phosphorylation sites on the serine/threonine kinase VRK1 Masahiko Negishi Characterization of adenylation on DNA Ligase IV Andrea Moon and Lars Pedersen Identification of bacterial pathogens by MALDI-MS David Kurtz Other projects that have been recently published, or have been submitted and accepted for publication include: With Dr. R.Scott Williams: Topoisomerase 2 (TOP2) DNA transactions are essential for life, and proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein crosslinks are resolved is unclear. Here, we show that the SUMO ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent Tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a "split-SIM" SUMO2 engagement platform. These findings uncover a ZATTTDP2 catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc. The specific contribution of the Mass Spectrometry Research and Support Group was to identify the proteins that immunoprecipitated with Tdp2. This resulted in the discovery of ZATT as a factor involved in DNA repair. With Dr. Robin Stanley: Las1 is a recently discovered endoribonuclease that collaborates with Grc3-Rat1-Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. We reported that the essential Las1 endoribonuclease requires it binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results established that Grc3 specifically directs Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively towards single-stranded RNA. Together Las1 and Grc3 assemble into a tetrameric complex that is required for competenent rRNA processing. The tetrameric Grc3/Las1 crosstalk draws unexpected parallels to endoribonucleases RNaseL and Ire1 and establishes Grc3/Las1 as a novel member of the RNaseL/Ire1 RNA Splicing Family. Together, our work provided mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a new example of a protein guided programmable endoribonuclease. The specific contribution of the Mass Spectrometry Research and Support Group was to characterize the recombinant proteins used in these experiments as well as to identify the cleavage site in the RNA substrate. Additional projects that have required more than negligible resources include efforts performed with the Blackshear, Cidlowski, Garantziotis, Hall, Hu, Kunkel, and Wilson laboratories.

在质谱研究和支持小组中，已经或正在进行各种蛋白质表征的服务和协作项目，其中大约4000个样本，分析了39位代表来自6个实验室分支机构和DNTP的主要研究人员或核心负责人的科学家。一项巨大的努力是支持蛋白质表达核心设施（PECF）和Bob Petrovich博士。 PMCF的作用是在将PECF将材料移交给使用者之前确认蛋白质水平的基因表达。仍在进行的其他已发表和未发表的项目包括：合作中的粉尘和蛋白质的特征 - 唐·库克（Don Cook），杰弗里·穆勒（Geoffrey Mueller）和罗伯特·伦敦蛋白质的表征：RNA加工酶中蛋白质交联 CRM1蛋白Raja Jothi的翻译后修饰的表征丝氨酸/苏氨酸激酶VRK1 Masahiko Negishi上磷酸化位点的表征 DNA连接酶IV Andrea Moon和Lars Pedersen上腺苷酸化的表征 MALDI-MS David Kurtz鉴定细菌病原体最近已发布或已提交并接受的其他项目包括： R.Scott Williams博士：拓扑异构酶2（TOP2）DNA交易对生命至关重要，并通过形成Top2裂解复合物（TOP2CC），这是一种共价酶-DNA反应中间体，该反应容易被有效的抗癌TOP2药物捕获。尚不清楚遗传毒性TOP2 DNA-蛋白交联的解析。在这里，我们表明Sumo连接酶Zatt（ZNF451）是一种多功能DNA修复因子，可控制对TOP2损伤的细胞反应。 Zatt与TOP2CC的结合促进了蛋白酶体独立的酪酶-DNA磷酸二酯酶2（TDP2）水解酶在失速的TOP2CC上的活性。 Zatt Sumo Gigase活动进一步促进了TDP2与Sumoypated Top2的相互作用，从而通过“拆分SIM” SUMO2参与平台调节有效的TDP2募集。这些发现发现了ZattTDP2催化和SUMO2调节的途径，用于直接分辨top2cc。质谱研究和支持组的具体贡献是鉴定用TDP2免疫沉淀的蛋白质。这导致发现Zatt是DNA修复涉及的一个因素。与Robin Stanley博士：LAS1是最近发现的内核核酸酶，它与GRC3-RAT1-RAI1合作处理前体核糖体RNA（RRNA），但其作用机理仍然未知。哺乳动物LAS1基因的破坏与先天性致命运动神经元疾病和X连锁智障疾病有关，因此强调了了解LAS1调节和功能的必要性。我们报道说，对于特定的C2裂解，必需的LAS1内核酸酶需要它结合伴侣，多核苷酸激酶GRC3。我们的结果表明，GRC3在体外和酿酒酵母中专门将LAS1内核酸酶切割到其靶向的C2位点。此外，我们观察到GRC3激酶活性仅针对单链RNA的LAS1依赖性激活。 LAS1和GRC3一起组装成竞争性rRNA处理所需的四聚体配合物。四聚体GRC3/LAS1串扰使意外的相似之处与内核核酸酶和IRE1相似，并建立了GRC3/LAS1作为RNASEL/IRE1 RNA剪接家族的新成员。总之，我们的工作为调节LAS1内核酸酶调节的机械见解提供了识别，并将四聚体GRC3/LAS1复合物确定为蛋白质引导的可编程内核酸酶的新例子。质谱研究和支持组的特定贡献是表征这些实验中使用的重组蛋白，以及鉴定RNA底物中的裂解位点。除了可以忽略不计的资源所需的其他项目还包括与Blackshear，Cidlowski，Garantziotis，Hall，Hu，Hu，Kunkel和Wilson Laboratories一起进行的努力。