Protein Microcharacterization

质谱研究和支持小组已经或正在进行蛋白质表征方面的各种服务和合作项目，分析了来自6个实验室分支和DNTP的23名主要研究人员或核心负责人的39名科学家的约4000个样品。 其中一个很大的努力是支持蛋白质表达核心设施（PECF）和鲍勃彼得罗维奇博士。PMCF的作用是在PECF将材料移交给其用户之前确认蛋白质水平的基因表达。 其他已公布和未公布的项目仍在进行中，包括： 来自过敏原的灰尘和蛋白质的表征- Don Cook，Geoffrey Mueller和Robert伦敦 蛋白质的表征：RNA加工酶中的蛋白质交联 Raja Jothi CRM 1蛋白翻译后修饰的表征 丝氨酸/苏氨酸激酶VRK 1磷酸化位点的表征 DNA连接酶IV腺苷酸化的表征 MALDI-MS鉴定细菌病原体大卫库尔茨 最近出版或已提交并接受出版的其他项目包括： 斯科特威廉姆斯博士：拓扑异构酶2（TOP2）DNA交换对于生命是必不可少的，并且通过形成TOP2切割复合物（TOP2 cc）进行，TOP2 cc是一种共价酶-DNA反应中间体，易于被有效的抗癌TOP2药物捕获。遗传毒性TOP2 DNA-蛋白质交联如何解决尚不清楚。在这里，我们表明，SUMO连接酶ZATT（ZNF 451）是一种多功能的DNA修复因子，控制TOP2损伤的细胞反应。ZATT与TOP2 cc的结合促进了停滞的TOP2 cc上的蛋白酶体非依赖性酪氨酸基-DNA磷酸二酯酶2（TDP 2）水解酶活性。ZATT SUMO连接酶活性进一步促进TDP 2与SUMO化TOP 2的相互作用，通过“split-SIM”SUMO 2接合平台调节有效的TDP 2募集。这些发现揭示了ZATTTDP 2催化和SUMO 2调节的直接解析TOP2 cc的途径。 质谱研究和支持小组的具体贡献是鉴定与Tdp 2免疫沉淀的蛋白质。 这导致了ZATT作为参与DNA修复的因子的发现。 Robin Stanley博士：Las 1是最近发现的核糖核酸内切酶，与Grc 3-Rat 1-Rai 1合作加工前体核糖体RNA（rRNA），但其作用机制尚不清楚。哺乳动物Las 1基因的破坏与先天性致死性运动神经元疾病和X连锁智力障碍有关，因此强调了了解Las 1调节和功能的必要性。 我们报道了必需的Las 1内切核糖核酸酶需要它的结合伙伴，多核苷酸激酶Grc 3，特异性C2切割。我们的研究结果表明，Grc 3特异性地指导Las 1内切核糖核酸酶切割其靶向C2位点在体外和酿酒酵母。 此外，我们观察到Las 1依赖性激活的Grc 3激酶活性专门对单链RNA。Las 1和Grc 3一起组装成一个四聚体复合物，这是有能力的rRNA加工所必需的。四聚体Grc 3/Las 1串扰与核糖核酸内切酶RNaseL和Ire 1产生了意想不到的相似之处，并将Grc 3/Las 1确立为RNaseL/Ire 1 RNA剪接家族的新成员。总之，我们的工作为Las 1核糖核酸内切酶的调节提供了机制上的见解，并将四聚体Grc 3/Las 1复合物鉴定为蛋白质引导的可编程核糖核酸内切酶的新实例。 质谱研究和支持小组的具体贡献是表征这些实验中使用的重组蛋白以及鉴定RNA底物中的切割位点。 其他需要超过可忽略不计的资源的项目包括与黑剪、Cidlowski、Garantziotis、Hall、Hu、Kunkel和Wilson实验室一起进行的工作。

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 Other published and unpublished projects that are still ongoing include: Characterization of dust and proteins from allergens in collaboration - Don Cook, Geoffrey Mueller, and Robert London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.