Ribosomal Proteins S27E, P2, and L37A from Marine Invertebrates

Single complementary DNAs encoding sequences for 40S ribosomal proteins related to S27E from the American lobster Homarus americanus and mussel Mytilus galloprovincialis were characterized. Single genes for ribosomal proteins L37A and P2 from the gumboot chiton Cryptochiton stellerii are similarly described. The lobster S27E protein contains the highly conserved cysteine residues, suggesting its likely designation in the C₄ protein family containing zinc finger motifs. The lobster S27E protein also appears to have an intermediate gene copy number between lower and higher euckaryotes. Expression of the S27E protein in lobster hepatopancreas was slightly elevated during several postmolt and premolt stages. Chlorinated pesticide treatment significantly reduced S27E expression in hepatopancreas, indicating that this gene is responsive to endogenous and exogenous cues. Received March 6, 1998; accepted October 2, 1998.     

Steady States and Oscillations in the p53/Mdm2 Network

p53 is activated in response to events compromising the genetic integrity of a cell. Recent data show that p53 activity does not increase steadily with genetic damage but rather fluctuates in an oscillatory fashion (Lahav et al., Nature Genetics, 36, 147-150, 2004). Theoretical studies suggest that oscillations can arise from a combination of positive and negative feedbacks or from a long negative feedback loop alone. Both negative and positive feedbacks are present in the p53/Mdm2 network, but it is not known what roles they play in the oscillatory response to DNA damage. We developed a mathematical model of p53 oscillations based on positive and negative feedbacks in the p53/Mdm2 network. According to the model, the system reacts to DNA damage by moving from a stable steady state into a region of stable limit cycles. Oscillations in the model are born with large amplitude, which guarantees an all-or-none response to damage. As p53 oscillates, damage is repaired and the system moves back to a stable steady state with low p53 activity. The model reproduces experimental data in quantitative detail. We suggest new experiments for dissecting the contributions of negative and positive feedbacks to the generation of oscillations.     

Ribosomal protein L37 and the p53 network

Comment on: Llanos S, et al. Cell Cycle 2010; 9:4005–2.     

Tissue-specific differences of p53 inhibition by Mdm2 and Mdm4

The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development.     

Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53

The product of the Mdm2 oncogene directly interacts with p53 and promotes its ubiquitination and proteasomal degradation. Initial biological studies identified nuclear export sequences (NES), similar to that of the Rev protein from the human immunodeficiency virus, both in Mdm2 and p53. The reported phenotypes resulting from mutation of these NESs, together with results obtained using the nuclear export inhibitor leptomycin B (LMB), have led to a model according to which nuclear export of p53 (via either the NES of Mdm2 or its own NES) is required for efficient p53 degradation. In this study we demonstrate that Mdm2 can promote degradation of p53 in the nucleus or in the cytoplasm, provided both proteins are colocalized. We also investigated if nuclear export is an obligate step on the p53 degradation pathway. We find that (1) when proteasome activity is inhibited, ubiquitinated p53 accumulates in the nucleus and not in the cytoplasm; (2) Mdm2 with a mutated NES can efficiently mediate degradation of wild type p53 or p53 with a mutated NES; (3) the nuclear export inhibitor LMB can increase the steady-state level of p53 by inhibiting Mdm2-mediated ubiquitination of p53; and (4) LMB fails to inhibit Mdm2-mediated degradation of the p53NES mutant, demonstrating that Mdm2-dependent proteolysis of p53 is feasible in the nucleus in the absence of any nuclear export. Therefore, given cocompartmentalization, Mdm2 can promote ubiquitination and proteasomal degradation of p53 with no absolute requirement for nuclear to cytoplasmic transport.     

miR-661 downregulates both Mdm2 and Mdm4 to activate p53

The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.     

Nucleolar Arf sequesters Mdm2 and activates p53

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

Conservation of all three p53 family members and Mdm2 and Mdm4 in the cartilaginous fish

Analysis of the genome of the elephant shark (Callorhinchus milii), a member of the cartilaginous fishes (Class Chondrichthyes), reveals that it encodes all three members of the p53 gene family, p53, p63 and p73, each with clear homology to the equivalent gene in bony vertebrates (Class Osteichthyes). Thus, the gene duplication events that lead to the presence of three family members in the vertebrates dates to before the Silurian era. It also encodes Mdm2 and Mdm4 genes but does not encode the p19^Arf gene. Detailed comparison of the amino acid sequences of these proteins in the vertebrates reveals that they are evolving at highly distinctive rates, and this variation occurs not only between the three family members but extends to distinct domains in each protein.     

Phospho-ser 15-p53 translocates into mitochondria and interacts with Bcl-2 and Bcl-xL in eugenol-induced apoptosis

Our previous studies demonstrated that antiallergic effects of herbs such as clove and Magnoliae Flos (MF) resulted from the induction of apoptosis in mast cells. We here examined whether the antiallergic activity was caused by eugenol (4-allyl-2-methoxyphenol) which was one of major ingredients in the essential oils or extracts of numerous plants including clove and Magnoliae Flos. RBL-2H3 cells were treated with eugenol, and DNA electrophoresis, Western blotting, immunocytochemistry, confocal microscopy and immunoprecipitation were conducted. Effect of eugenol was tested using a rat anaphylaxis model. RBL-2H3 cells treated with eugenol showed typical apoptotic manifestations and translocation of p53 into mitochondria. Antisense p53 partially prevented the induction of apoptosis. Noticeably, we observed that p53 translocated into mitochondria was phosphorylated on ser 15. Phospho-ser 15-p53 physically interacted with Bcl-2 and Bcl-xL in mitochondria and its translocation into mitochondria preceded cytochrome c release and mitochondrial membrane potential (MMP) reduction. We also depicted that the survival of animals even after administration of the fatal dose of compound 48/80 might result from the decreased number of mast cells by eugenol pretreatment. In conclusion, eugenol induces apoptosis in mast cells via translocation of phospho-ser 15-p53 into mitochondria.