Regulation and function of mitochondrial Hep27: a novel modulator of the Mdm2-p53 pathway

The ever-expanding role of p53 in cellular metabolism, apoptosis, and cell cycle control has led to increasing interest in defining the stress response pathways that regulate Mdm2. In an effort to identify novel Mdm2 binding partners, we performed a large-scale immunoprecipitation of Mdm2 in the osteosarcoma U2OS cell line. One significant binding protein identified was Hep27, a member of the short-chain alcohol dehydrogenase/reductase (SDR) family of enzymes. Here we demonstrate the Hep27 pre-protein contains an N-terminal mitochondrial targeting signal that is cleaved following mitochondrial import, resulting in mitochondrial matrix accumulation of mature Hep27. A fraction of mitochondrial Hep27 translocates to the nucleus, where it binds to Mdm2 in the central domain, resulting in attenuation of Mdm2 mediated p53 degradation. In addition, Hep27 is regulated at the transcriptional level by the proto-oncogene c-Myb and is required for c-Myb induced p53 activation. Breast cancer gene expression analysis correlated estrogen receptor (ER) status with Hep27 expression and p53 function, providing a potential in vivo link between estrogen receptor signaling and p53 activity. Our data demonstrate a unique ER-c-Myb-Hep27-Mdm2-p53 mitochondria-to-nucleus signaling pathway that may have functional significance for ER positive breast cancers

Development of a bioprinter-based method for incorporating metabolic competence into high-throughput in vitro assays

The acceptance and use of in vitro data for hazard identification, prioritization, and risk evaluation is partly limited by uncertainties associated with xenobiotic metabolism. The lack of biotransformation capabilities of many in vitro systems may under- or overestimate the hazard of compounds that are metabolized to more or less active metabolites in vivo. One approach to retrofitting existing bioassays with metabolic competence is the lid-based Alginate Immobilization of Metabolic Enzymes (AIME) method, which adds hepatic metabolism to conventional high-throughput screening platforms. Here, limitations of the lid-based AIME method were addressed by incorporating bioprinting, which involved depositing S9-encapsulated microspheres into standard 384-well plates with requisite cofactors for phase I and II hepatic metabolism. Objectives of this study included: 1) compare the lid-based and AIME bioprinting methods by assessing the enzymatic activity of a common cytochrome P450 (CYP) enzyme, 2) use biochemical assays with the bioprinting method to characterize additional measures of phase I and II metabolic activity, and 3) evaluate the bioprinting method by screening 25 chemicals of known metabolism-dependent bioactivity in the VM7Luc estrogen receptor transactivation (ERTA) assay. A comparison of the two methods revealed comparable precision and dynamic range. Activity of additional CYP enzymes and glucuronidation was observed using the AIME bioprinting method. The ERTA experiment identified 19/21 ER-active test chemicals, 14 of which were concordant with expected biotransformation effects (73.7%). Additional refinement of the AIME bioprinting method has the potential to expand high-throughput screening capabilities in a robust, accessible manner to incorporate in vitro metabolic competence

p53-inducible DHRS3 Is an Endoplasmic Reticulum Protein Associated with Lipid Droplet Accumulation

The transcription factor p53 plays a critical role in maintaining homeostasis as it relates to cellular growth, proliferation, and metabolism. In an effort to identify novel p53 target genes, a microarray approach was utilized to identify DHRS3 (also known as retSDR1) as a robust candidate gene. DHRS3 is a highly conserved member of the short chain alcohol dehydrogenase/reductase superfamily with a reported role in lipid and retinoid metabolism. Here, we demonstrate that DHRS3 is an endoplasmic reticulum (ER) protein that is shuttled to the ER via an N-terminal endoplasmic reticulum targeting signal. One important function of the ER is synthesis of neutral lipids that are packaged into lipid droplets whose biogenesis occurs from ER-derived membranes. DHRS3 is enriched at focal points of lipid droplet budding where it also localizes to the phospholipid monolayer of ER-derived lipid droplets. p53 promotes lipid droplet accumulation in a manner consistent with DHRS3 enrichment in the ER. As a p53 target gene, the observations of Dhrs3 location and potential function provide novel insight into an unexpected role for p53 in lipid droplet dynamics with implications in cancer cell metabolism and obesity

p53 upregulates PLCε-IP3-Ca2+ pathway and inhibits autophagy through its target gene Rap2B

The tumor suppressor p53 plays a pivotal role in numerous cellular responses as it regulates cell proliferation, metabolism, cellular growth, and autophagy. In order to identify novel p53 target genes, we utilized an unbiased microarray approach and identified Rap2B as a robust candidate, which belongs to the Ras-related GTP-binding protein superfamily and exhibits increased expression in various human cancers. We demonstrated that p53 increases the intracellular IP3 and Ca2+ levels and decreases the LC3 protein levels through its target gene Rap2B, suggesting that p53 can inhibit the autophagic response triggered by starvation via upregulation of the Rap2B-PLCε-IP3-Ca2+ pathway. As a confirmed target gene of p53, we believe that further investigating potential functions of Rap2B in autophagy and tumorigenesis will provide a novel strategy for cancer therapy

An ARF-Independent c-MYC-Activated Tumor Suppression Pathway Mediated by Ribosomal Protein-Mdm2 Interaction

In vitro studies have shown that inhibition of ribosomal biogenesis can activate p53 through ribosomal protein (RP)-mediated suppression of Mdm2 E3 ligase activity. To study the physiological significance of the RP-Mdm2 interaction, we generated mice carrying a cancer-associated cysteine-to-phenylalanine substitution in the zinc finger of Mdm2 that disrupted its binding to RPL5 and RPL11. Mice harboring this mutation, although retain normal p53 response to DNA damage, lack p53 response to perturbations in ribosome biogenesis. Loss of RP-Mdm2 interaction significantly accelerates Eμ-Myc induced lymphomagenesis. Furthermore, ribosomal perturbation induced p53 response does not require tumor suppressor p19Arf. Collectively, our findings establish RP-Mdm2 interaction as a genuine p53 stress-signaling pathway activated by aberrant ribosomal biogenesis and essential for safeguarding against oncogenic c-Myc-induced tumorigenesis

An ARF-Independent c-MYC-Activated Tumor Suppression Pathway Mediated by Ribosomal Protein-Mdm2 Interaction

In vitro studies have shown that inhibition of ribosomal biogenesis can activate p53 through ribosomal protein (RP)-mediated suppression of Mdm2 E3 ligase activity. To study the physiological significance of the RP-Mdm2 interaction, we generated mice carrying a cancer-associated cysteine-to-phenylalanine substitution in the zinc finger of Mdm2 that disrupted its binding to RPL5 and RPL11. Mice harboring this mutation, although retain normal p53 response to DNA damage, lack p53 response to perturbations in ribosome biogenesis. Loss of RP-Mdm2 interaction significantly accelerates Eμ-Myc induced lymphomagenesis. Furthermore, ribosomal perturbation induced p53 response does not require tumor suppressor p19Arf. Collectively, our findings establish RP-Mdm2 interaction as a genuine p53 stress-signaling pathway activated by aberrant ribosomal biogenesis and essential for safeguarding against oncogenic c-Myc-induced tumorigenesis

Technical Evaluation and Standardization of the Human ThyroidMicrotissue Assay

Presentation to the US EPA and Japan Ministry of the Environment16th Bilateral Meeting on Endocrine Disruption Test Methods Development September 2023Search for CCTE records in EPA’s Science Inventory by typing in the title at this link.https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25</p

Evaluating the Human Relevance of Potential Thyroid Disrupting Chemicals Using In vitro New Approach Methods

Presentation to the Society of Toxicology 62nd Annual Meeting and ToxExpo March 2023 Search for CCTE records in EPA’s Science Inventory by typing in the title at this link. https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25 </p

Development of a Human Thyroid Microtissue Model for Evaluation of Thyroid Hormone Synthesis

Presentation at RIVM (Netherlands National Institute for Public Health and the Environment) visit to EPA April 201

The Ribosomal Protein-Mdm2-p53 Pathway and Energy Metabolism: Bridging the Gap between Feast and Famine

Cellular growth and division are two fundamental processes that are exquisitely sensitive and responsive to environmental fluctuations. One of the most energetically demanding functions of these processes is ribosome biogenesis, the key component to regulating overall protein synthesis and cell growth. Perturbations to ribosome biogenesis have been demonstrated to induce an acute stress response leading to p53 activation through the inhibition of Mdm2 by a number of ribosomal proteins. The energy status of a cell is a highly dynamic variable that naturally contributes to metabolic fluctuations, which can affect both the rates of ribosome biogenesis and p53 function. This, in turn, determines whether a cell is in an anabolic, growth-promoting state or a catabolic, growth-suppressing state. Here the authors integrate the known functions of p53 to postulate how changes in nutrient availability may induce the ribosomal protein–Mdm2-p53 signaling pathway to modulate p53-dependent metabolic regulation