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. ? ? ? ? ?
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