Biology subjects

Doan, M. T.

Publications and source records attributed to Doan, M. T..

7 recordsLinked to original sources

bioRxiv2021

O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation

Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote tumor growth. O-GlcNAcylation has been shown to be elevated by increasing O-GlcNAc transferase (OGT) in many cancers and reduced O-GlcNAcylation can block cancer growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA production by regulating phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin-dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that ACSS2 Ser-267 phosphorylation regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Importantly, we show that pharmacologically targeting OGT and CDK5 reduces GBM growth ex vivo. Thus, the OGT/CDK5/ACSS2 pathway may be a way to target altered metabolic dependencies in brain tumors.

cancer biology↗

bioRxiv2020

Cumulus cell acetyl-CoA metabolism from acetate is associated with maternal age but only partially with oocyte maturity

Cumulus cell (CC) clumps that associate with oocytes provide the oocytes with growth and signaling factors. Thus, the metabolism of the CCs may influence oocyte function and CC metabolism may be predictive of oocyte competence for in vitro fertilization. CCs are thought to be highly glycolytic but data on other potential carbon substrates are lacking in humans. This was a prospective and blinded cohort study that was designed to examine the substrate utilization of CCs by age and oocyte competence. Individual sets of CC clumps from participants were removed after oocyte retrieval procedure, incubated with stable isotope labeled substrates, and analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for isotopologue enrichment of major metabolic intermediates, including acetyl-CoA. The acyl-chain of acetyl-CoA contains 2 carbons that can be derived from 13C-labeled substrates resulting in a M+2 isotopologue that contains 2 13C atoms. Comparing the fate of three major carbon sources, mean enrichment of M+2 acetyl-CoA (mean, standard deviation) was for glucose (3.6, 7.7), for glutamine (9.4, 6.2), and for acetate (20.7, 13.9). Due to this unexpected high and variable labeling from acetate, we then examined acetyl-CoA mean % enrichment from acetate of in 278 CCs from 21 women [≤]34 (49.06, 12.73) decreased with age compared to 124 CCs from 10 women >34 (43.48, 16.20) (p=0.0004, t test). The CCs associated with the immature prophase I oocytes had significantly lower enrichment in M+2 acetyl CoA compared to the CCs associated with the metaphase I and metaphase II oocytes (difference: -6.02, CI: -1.74,-13.79, p=0.013). Acetate metabolism in individual CC clumps was positively correlated with oocyte maturity and decreased with maternal age. These findings indicate that CC metabolism of non-glucose substrates should be investigated relative to oocyte function and age-related fertility.

biochemistry↗

bioRxiv2019

Direct anabolic three carbon metabolism to a six carbon metabolite occurs in human heart and in vivo across mouse tissues

Anabolic metabolism of carbon in mammals is mediated via the one and two carbon carriers S-adenosyl methionine and acetyl-coenzyme A (acetyl-CoA). In contrast, anabolic metabolism using three carbon units via propionate is not thought to occur. Mammals are primarily thought to oxidize the 3-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. We found that this may not be absolute and that in mammals one non-oxidative fate of two units of propionyl-CoA is to condense to a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this pathway using purified protein extracts provided limited substrates and confirmed the product with a synthetic standard. In whole-body in vivo stable isotope tracing with infusion of 13C-labeled valine achieving steady state, 2M2PE-CoA formed via propionyl-CoA in multiple murine tissues including heart, kidney, and to a lesser degree in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three to six carbon reaction conserved in humans and mice that utilizes three carbons via propionate. Highlights- Synthesis and confirmation of structure 2-methyl-2-pentenoyl-CoA - In vivo fate of valine across organs includes formation of a 6-carbon metabolite from propionyl-CoA - Ex vivo metabolism of propionate in the human heart includes direct anabolism to a 6-carbon product - In both cases, this reaction occurred at physiologically relevant concentrations of propionate and valine - In vitro this pathway requires propionyl-CoA and NADH/NADPH as substrates

biochemistry↗

bioRxiv2019

Subcellular metabolic pathway kinetics are revealed by correcting for artifactual post harvest metabolism

OBJECTIVEThe dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of 13C-labeled substrates targeting central metabolic pathways.\n\nMETHODSWe designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS).\n\nRESULTSWe show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic, and reflects the metabolic capacity of the sub-cellular compartment analyzed; but is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondrial and cytosolic metabolic analyses.\n\nCONCLUSIONSOur data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment.

biochemistry↗

bioRxiv2018

Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry

Quantification of cellular deoxyribonucleoside mono-(dNMP), di-(dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.

biochemistry↗

bioRxiv2018

Suppression of p16 increases nucleotide synthesis via mTORC1

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurred via activation of mTORC1 signaling, which directly mediated increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlated with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibited proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.\n\nHighlightsO_LImTORC1 is activated by p16 knockdown to increase nucleotide synthesis and bypass senescence\nC_LIO_LImTORC1 directly increases translation RPIA to increase ribose-5-phosphate\nC_LIO_LIActivation of mTORC1 pathway downstream of p16 suppression is independent of RB\nC_LIO_LIRPIA suppression induces senescence only in cells and tumors with low p16\nC_LI

cancer biology↗

bioRxiv2018

Identification and Quantification of Paclitaxel and its Metabolites in Human Meconium from Newborns with Gestational Chemotherapeutic Exposure

ObjectiveCancer diagnosis during pregnancy occurs in 1 out of 1000 pregnancies with common malignancies including breast and hematological cancers. Fetal exposure to currently utilized agents is poorly described. We directly assessed fetal exposure by screening meconium from 23 newborns whose mothers had undergone treatment for cancer during pregnancy.\n\nStudy DesignMeconium was collected from newborns whose mothers were diagnosed with cancer during pregnancy and underwent chemotherapy in the second or third trimester as part of the Cancer and Pregnancy Registry. We conducted screening of 23 meconium samples for chemotherapeutics and known metabolites of chemotherapeutics by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Putative identification of paclitaxel and/or its metabolites was made in 8 screened samples. In positively screened samples, we quantified paclitaxel, 3-p-hydroxypaclitaxel, and 6-hydroxypaclitaxel by stable isotope dilution-LC-HRMS.\n\nResultsMean levels of paclitaxel were 399.9 pg/mg in meconium samples from newborn born to mothers that underwent chemotherapy during pregnancy. 3-p-hydroxypaclitaxel and 6-hydroxypaclitaxel mean levels were 105.2 and 113.4 pg/mg meconium, respectively.\n\nConclusionIntact paclitaxel, and at least two of its major metabolites were detected in meconium, providing unambiguous confirmation of human fetal exposure. Variability in meconium levels between individuals may indicate a potential for reducing fetal exposure based on timing, dosing, and individual characteristics. This preliminary study may provide an efficient approach for examining the effects of cancer diagnosis during pregnancy on other outcomes by providing a measure of direct fetal exposure.

pharmacology and toxicology↗