The role of L5, L11 and L23 in the MDM2-p53 feedback regulation

L5、L11 和 L23 在 MDM2-p53 反馈调节中的作用

• 批准号: 7888287
• 负责人: Hua Lu
• 金额: $ 25.66万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888287
• 关键词: Address; Animals; Binding; Biochemical; Biogenesis; Biological; Cell Cycle Regulation; Cells; Coupling; DNA-Directed RNA Polymerase; Dactinomycin; Dose; Drug Design; EP300 gene; Equilibrium; Feedback; Fibroblasts; Funding; G1 Arrest; G1 Phase; Genes; Goals; Human; In Vitro; Incidence; Individual; Kinetics; Lead; Link; MDM2 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Molecular; Mus; Mutagenesis; Mutation; NPM1 gene; PCAF gene; Pathway interactions; Peptides; Phosphorylation; Physiological; Play; Process; Protein Binding Domain; Protein Biosynthesis; Protein Subunits; Proteins; Proteolysis; Regulation; Reporting; Research; Ribosomal Proteins; Ribosomal RNA; Role; Signal Transduction; Stimulus; Stress; System; TP53 gene; Testing; Tumor Suppressor Proteins; Ubiquitination; Work; Zebrafish; antitumor drug; base; cancer cell; cell growth; extracellular; in vitro activity; peptide L; research study; response; tumor; tumorigenesis

DESCRIPTION (provided by applicant): The long term goal of this project is to elucidate the molecular and biochemical mechanisms underlying the regulation of the MDM2-p53 autoregulatory feedback loop in response to physiological and pathological stimuli. Because the malfunction of the MDM2-p53 feedback loop is associated with the majority of human cancers, this loop is subjected to tight regulation by a variety of intracellular and extracellular signals. In an attempt to understand the fundamental molecular basis of the regulation, our previously funded research has focused on dissecting the involvement of p300/CBP and PCAF in this loop. Most of the aims of that proposal have been completed in my lab, and some questions raised in the previous proposal have also been addressed by other groups, over the past 3 years. While working on this project, we have also attempted to identify cellular MDM2 regulators. To our surprise, three ribosomal large (L) 60S subunit proteins, L5, L11 and L23, have been found to activate p53 by associating with MDM2 and inhibiting its function on p53 degradation in response to actinomycin D-induced ribosomal stress. Likewise, two earlier reports have also shown that L11 induces p53 by repressing MDM2 function in response to ribosomal stress. Ribosomal stress occurs when ribosomal biogenesis, including rRNA synthesis, rRNA processing and ribosomal assembly, is perturbed, e.g., a low dose (<5 nM) of actinomycin D can cause ribosomal stress by specifically inhibiting RNA polymerase l-catalyzed rRNA synthesis. Increasing evidence supports the idea that p53 is also a major player in coupling ribosomal biogenesis with cell cycle control. p53 is activated to arrest cells at G1 phase through a phosphorylation-independent mechanism in response to various ribosomal stresses. Our findings link the L proteins with the ribosomal stress-p53 pathway and suggest a potential role for these proteins in tumorigenesis. Consistently, others have shown that heterozygous mutations of 11 individual ribosomal protein genes in zebrafish are associated with high tumor incidence. Also others shown that the tumor suppressor ARF, which has been shown to activate p53 by directly inhibiting MDM2 function, suppresses rRNA processing by mediating proteasomal turnover of the rRNA processor B23. This result suggests that ARF may also activate p53 by causing ribosomal stress. These studies lead to an important hypothesis that ribosomal L5, L11 and L23 proteins, besides their essential role in de novo protein synthesis, may also play a crucial role in p53 response to ribosomal stress. This also raises the question of how exactly these L proteins regulate the MDM2-p53 loop. To test this hypothesis and to address this question, three specific aims are proposed in this application as follows: 1). To elucidate the biochemical mechanisms underlying the inhibition of MDM2 function by the L proteins; 2). To identify small MDM2-binding L protein-derived peptides for p53 activation; 3). To determine the role of L11 in the ARF-MDM2-p53 pathway. Achieving these aims using biochemical, biophysical, cellular and molecular biological approaches would not only help us better understand the molecular mechanisms that govern p53 activation and cell growth control in response to ribosomal stress, but also provide useful information for identifying small peptide molecules that target MDM2 as potential anti-tumor drugs for pharmacological study.

描述（由申请人提供）：本项目的长期目标是阐明MDM 2-p53自动调节反馈环在生理和病理刺激下调节的分子和生化机制。由于MDM 2-p53反馈环的功能障碍与大多数人类癌症相关，因此该环受到各种细胞内和细胞外信号的严格调节。为了了解调控的基本分子基础，我们以前资助的研究集中在解剖p300/CBP和PCAF在这个循环中的参与。该提案的大部分目标已经在我的实验室完成，在过去的3年里，其他小组也解决了前一个提案中提出的一些问题。在进行这个项目的同时，我们还试图鉴定细胞MDM 2调节剂。令人惊讶的是，已经发现三种核糖体大（L）60 S亚基蛋白L5、L11和L23通过与MDM 2结合并抑制其对p53降解的功能而激活p53，这是对放线菌素D诱导的核糖体应激的响应。同样，两个早期的报道也表明，L11通过抑制响应于核糖体应激的MDM 2功能来诱导p53。当核糖体生物发生（包括rRNA合成、rRNA加工和核糖体组装）受到干扰时，发生核糖体应激，例如，低剂量（<5 nM）的放线菌素D可通过特异性抑制RNA聚合酶I催化的rRNA合成而引起核糖体应激。越来越多的证据支持p53也是核糖体生物合成与细胞周期控制耦合的主要参与者的观点。p53被激活，通过磷酸化非依赖性机制将细胞阻滞在G1期，以响应各种核糖体应激。我们的研究结果与核糖体应激p53通路的L蛋白，并建议这些蛋白质在肿瘤发生的潜在作用。与此相一致，其他人已经表明，斑马鱼中11个个体核糖体蛋白基因的杂合突变与高肿瘤发病率相关。其他人也表明，肿瘤抑制因子ARF（已显示通过直接抑制MDM 2功能激活p53）通过介导rRNA处理器B23的蛋白酶体周转来抑制rRNA处理。这一结果表明，ARF也可能通过引起核糖体应激激活p53。这些研究导致了一个重要的假设，核糖体L5，L11和L23蛋白，除了它们在从头蛋白质合成中的重要作用，也可能在p53对核糖体应激的反应中发挥至关重要的作用。这也提出了一个问题，即这些L蛋白究竟如何调节MDM 2-p53环。为了测试该假设并解决该问题，在本申请中提出了如下三个具体目标：1）。阐明L蛋白抑制MDM 2功能的生化机制; 2）.鉴定用于p53活化的小MDM 2结合L蛋白衍生肽; 3）.确定L11在ARF-MDM 2-p53通路中的作用。利用生物化学、生物物理学、细胞生物学和分子生物学方法实现这些目标，不仅有助于我们更好地理解核糖体应激时p53激活和细胞生长控制的分子机制，而且还为鉴定靶向MDM 2的小肽分子作为药理学研究的潜在抗肿瘤药物提供有用的信息。