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

描述(由申请人提供)：本项目的长期目标是阐明MDM2-P53自动调节反馈回路对生理和病理刺激的反应的分子和生化机制。因为MDM2-P53反馈环的故障与大多数人类癌症有关，所以这个环受到各种细胞内和细胞外信号的严格调控。为了了解该调控的基本分子基础，我们之前资助的研究重点是剖析p300/CBP和PCAF在这个环中的参与。在过去的三年里，该提案的大部分目标已经在我的实验室完成，之前提案中提出的一些问题也得到了其他小组的解决。在致力于这个项目的同时，我们还试图确定细胞MDM2调节器。令我们惊讶的是，已发现三个核糖体大(L)60S亚单位蛋白L5、L11和L23通过与MDM2结合并抑制其在放线菌素D诱导的核糖体应激反应中对P53降解的作用来激活P53。同样，早期的两份报告也表明，L11通过抑制核糖体应激反应中的MDM2功能来诱导p53。核糖体应激发生在核糖体的生物发生，包括rRNA合成、rRNA加工和核糖体组装受到干扰时，例如，低剂量(1；5 nM)的放线菌素D可以通过特异性地抑制核糖体聚合酶L催化的rRNA合成而引起核糖体应激。越来越多的证据支持这样的观点，即p53也是将核糖体生物发生与细胞周期控制结合起来的主要参与者。P53被激活，通过一种不依赖于磷酸化的机制使细胞停滞在G1期，以响应各种核糖体应激。我们的发现将L蛋白与核糖体应激-P53途径联系起来，并提示这些蛋白在肿瘤发生中的潜在作用。其他研究人员一直表明，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有关。也有研究表明，肿瘤抑制因子ARF通过直接抑制MDM2功能来激活p53，通过介导rRNA处理器B23的蛋白酶体周转来抑制rRNA的加工。这一结果表明，ARF也可能通过引起核糖体应激而激活P53。这些研究导致了一个重要的假设，即核糖体L5、L11和L23蛋白除了在从头合成蛋白质中起重要作用外，还可能在核糖体应激反应中发挥关键作用。这也提出了一个问题，即这些L蛋白到底是如何调控mdm2-p53环的。为了验证这一假设并解决这一问题，在本申请中提出了三个具体目标：1)。阐明L蛋白抑制MDM2功能的生化机制；鉴定与MDM2结合的L蛋白衍生肽对P53的激活作用；3)探讨L11在ARF-MDM2-P53通路中的作用。利用生化、生物物理、细胞和分子生物学方法实现这些目标，不仅有助于我们更好地了解核糖体应激反应中调控P53激活和细胞生长控制的分子机制，而且还将为识别靶向MDM2的小肽分子作为潜在的抗肿瘤药物提供有用的信息。

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.