Growth Control Regulated by p53 and MDM2

亨特学院（隶属于纽约城市大学）的Jill Bargonetti获得了一笔资助，用于开展“p53和MDM2调控的生长控制”项目。Bargonetti实验室有证据表明，在某些细胞中可能存在功能失调的p53- mdm2反馈回路，当这种情况发生时，尽管形成了核p53- mdm2复合物，但p53并不是降解的目标。在具有这种功能失调的p53- mdm2复合物的细胞中，p53不会激活一些促进生长停滞的靶基因（如p21），但会激活其他有助于细胞存活的靶基因（如mdm2）。这项资助的目的是确定与功能失调的野生型p53相关的特定p53蛋白复合物是否实际上是p53- mdm2复合物；如果这种蛋白质复合物导致p53与核染色质中的mdm2基因相互作用。目的一是鉴定MDM2免疫反应蛋白的氨基酸序列，该蛋白特异性地与人类细胞系中功能失调的野生型p53和功能性p53共免疫沉淀。Bargonetti实验室发现了一种潜在的新型MDM2与功能失调的野生型p53相关。目的二是确定p53:MDM2蛋白复合物是否可以与核染色质中MDM2 p53结合位点相互作用。染色质免疫沉淀- pcr （CHIP-PCR）利用p53特异性和MDM2特异性单克隆抗体，用于研究p53和MDM2与MDM2启动子区域（P1和P2）的核“体内”相互作用。目标三是确定具有功能性反馈回路的细胞系与上述功能失调的细胞系中p53和MDM2的mRNA序列和磷酸化状态。来自功能失调的野生型p53细胞系的RT-PCR mdm2产物以及来自功能正常的p53细胞系的产物正在测序中。巢式聚合酶链反应被用于比较细胞系中功能失调的野生型p53与功能正常的p53的剪接mdm2转录本，以观察是否存在差异。磷酸化特异性抗体用于研究p53的磷酸化状态。亨特学院是一所少数族裔大学。本科生被招募到实验室并在实验室接受指导，纽约城市大学研究生中心的研究生是Bargonetti研究团队的积极成员。正在进行的研究关注的是p53的调控。p53是一种细胞蛋白，作为中央控制器接收来自许多不同途径的信号，并控制许多下游途径。信号转导通路必须利用p53作为主要连接点，就像来自许多不同目的地的旅行者在出发前往不同目的地之前在主要车站穿过路径一样。p53- mdm2反馈回路是p53的一个关键信号通路，即p53开启mdm2，而mdm2蛋白使p53失活。正在进行的研究是为了检查p53-MDM2反馈回路的功能障碍。这两种蛋白相互作用，但相互作用后的信号不抑制细胞生长。目前正在研究这种功能失调循环的机制。Bargonetti实验室强调科学的卓越性，学生们在数据的专业展示、科学合作、团队合作和道德方面受到指导。这个项目有可能确定一种由MDM2调控p53的新形式，同时培养新一代的科学家。

A grant has been awarded to Jill Bargonetti at Hunter College (part of the City University of New York) to carry out the project "Growth Control Regulated by p53 and MDM2". The Bargonetti laboratory has evidence that a dysfunctional p53-MDM2 feedback loop may exist in some cells and when this occurs p53 is not targeted for degradation despite the formation of a nuclear p53-MDM2 complex. In cells with such a dysfunctional p53-MDM2 complex, p53 does not activate some target genes (for example p21) that promote growth arrest, but does activate other gene targets (like mdm2) that aid in cell survival. The aims of this grant are to identify if a specific p53 protein complex that correlates with dysfunctional wild-type p53 is, in fact, a p53-MDM2 complex; and if this protein complex causes p53 to constitutively interact with the mdm2 gene in nuclear chromatin. Aim one is to identify the amino acid sequences of the MDM2 immuno-reactive proteins that specifically co-immunoprecipitate with dysfunctional wild-type p53 and functional p53 from human cell lines. The Bargonetti laboratory has found a potentially novel form of MDM2 associated with dysfunctional wild-type p53. Aim two is to determine if a p53:MDM2 protein complex can interact with the mdm2 p53-binding site in nuclear chromatin. Chromatin immuno-precipitation-PCR (CHIP-PCR), utilizing both p53 specific and MDM2 specific monoclonal antibodies, is used to investigate the nuclear "in vivo" interaction of p53 and MDM2 with the mdm2 promoter regions (P1 and P2). Aim three is to determine the mRNA sequences and phosphorylation states of p53 and MDM2 in cell lines that have a functional feedback loop versus those that are dysfunctional as described above. The RT-PCR mdm2 products from a cell line with dysfunctional wild-type p53 as well as products from a cell line with properly functioning p53 are being sequenced. Nested PCR is being used to compare the spliced mdm2 transcripts in the cell line with dysfunctional wild-type p53 to normally functioning p53 to see if there is a difference. Phospho-specific antibodies are used to investigate the phosphorylation state of p53. Hunter College is an undergraduate minority institution. Undergraduates are recruited into and mentored in the laboratory and graduate students from the Graduate Center of the City University of New York are active members of the Bargonetti research team. The studies being carried out concern the regulation of p53, a cellular protein that acts as a central controller receiving signals from many different routes and controlling numerous downstream pathways. Signal transduction pathways must utilize p53 as a major connection point in much the same way as travelers from many different destinations cross paths at major stations before embarking to different destinations. The p53-MDM2 feedback loop, whereby p53 turns on mdm2 and the MDM2 protein inactivates p53, is a key signaling pathway to p53. The studies being carried out are to examine a dysfunction p53-MDM2 feedback loop. The two proteins interact but the signal after interaction does not inhibit cell growth. The mechanism for this dysfunctional loop is being examined. Scientific excellence is stressed in the Bargonetti laboratory and the students are instructed in professional presentation of their data, scientific collaboration, collegiality and ethics. This project has the potential to identify a novel form of regulation of p53 by MDM2, while producing a new generation of scientists.