Mammalian Cell Metabolism Research Articles

Exosomal Nrf2: From anti-oxidant and anti-inflammation response to wound healing and tissue regeneration in aged-related diseases

Accumulation of oxidative stress in cells is an essential feature of cellular senescence and aging. This phenomenon is involved in different age-related diseases through dysregulation of homeostasis and impairing repair and regeneration (wound healing) capacity, which can suppress antioxidant responses such as the activity of antioxidant enzymes and damaged protein clearance system. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor which regulates basal and inducible expression pattern of specific genes (antioxidants and detoxifications) through antioxidant element response (ARE) sites in the stress condition, specifically in chronic and age-related stresses. Nrf2 maintains cellular redox hemostasis and promotes rejuvenation. Exosomes are nanoscale vesicles that are released by various cells to actively regulate the complex cellular signaling networks. Exosomal-Nrf2 and exosomal-Nrf2-mediated products can modulate oxidative hemostasis in target cells to induce tissue repairing with therapeutic proposes, and regeneration capability. In this study, we summarized the role of exosomal-Nrf2 in different age-related diseases, including diabetic foot ulcers, atherosclerosis, chronic heart failure, reproductive cell failures, and neurodegenerative diseases. In addition, we briefly explained the crosstalk between plant exosomes and mammalian cell metabolism in the benefit of cellular stress suppression.

Dissecting Mammalian Cell Metabolism through 13C- and 2H-Isotope Tracing: Interpretations at the Molecular and Systems Levels

Isotopic tracers have been widely used to probe specific pathways within metabolic networks. Tracers are metabolized through various metabolic routes, generating differential labeling patterns. Cor...

ATP Mimics pH-Dependent Dual Peroxidase-Catalase Activities Driving H 2 O 2 Decomposition

Adenosine triphosphate (ATP) is produced mainly in the mitochondrion, and its primary task is to function as a ubiquitous energy currency to meet the cellular metabolic demands in biological system...

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures.

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell‐based and indicator cell‐based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real‐time decision making during the biomanufacturing process. To support real‐time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell‐based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single‐use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read‐out from a traditional method.

Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Specific heme binding to heme regulatory motifs in iron regulatory proteins and its functional significance

Iron regulatory proteins (IRPs) control iron metabolism in mammalian cells by binding to the iron-responsive element (IRE) in the target mRNA. Heme regulatory motifs (HRMs) are conserved in the two IRP homologues IRP1 and IRP2 that specifically bind to two and three heme equivalents, respectively; however, only the heme binding to the iron-dependent degradation (IDD) domain of IRP2 causes heme-mediated oxidation, which does not occur in IRP1. Therefore, the functional significance of conserved HRMs outside the IDD domain is yet unclear. In this study, spectroscopic heme titration with IRP mutants confirmed heme binding to each HRM in IRPs, and the effect of heme binding to HRMs on IRE binding was examined. Native polyacrylamide gel electrophoresis analysis revealed that heme binding to HRMs near the IRE binding site inhibits complex formation between IRPs and IRE without oxidative modification, indicating that the function of HRMs varies outside and within the IDD domain. However, the formation of a typical reactive oxygen species (ROS), hydrogen peroxide, was spectroscopically detected in both heme-bound IRPs. Comparing the heme environmental structures surrounding HRMs, the flexible conformation and many amino acid residues sensitive to ROS of the IDD domain were suggested to promote specific oxidation by the generated hydrogen peroxide. Thus, heme binding to HRM near the IRE binding site sterically interferes with IRE binding, while HRM in the IDD domain facilitates specific heme-mediated oxidation of the protein moiety and the protein degradation via the ubiquitin-proteasome system, resulting in the inhibition of IRE binding.

Rat liver folate metabolism can provide an independent functioning of associated metabolic pathways

Folate metabolism in mammalian cells is essential for multiple vital processes, including purine and pyrimidine synthesis, histidine catabolism, methionine recycling, and utilization of formic acid. It remains unknown, however, whether these processes affect each other via folate metabolism or can function independently based on cellular needs. We addressed this question using a quantitative mathematical model of folate metabolism in rat liver cytoplasm. Variation in the rates of metabolic processes associated with folate metabolism (i.e., purine and pyrimidine synthesis, histidine catabolism, and influxes of formate and methionine) in the model revealed that folate metabolism is organized in a striking manner that enables activation or inhibition of each individual process independently of the metabolic fluxes in others. In mechanistic terms, this independence is based on the high activities of a group of enzymes involved in folate metabolism, which efficiently maintain close-to-equilibrium ratios between substrates and products of enzymatic reactions.

The Fate of Glutamine in Human Metabolism. The Interplay with Glucose in Proliferating Cells.

Genome-scale models of metabolism (GEM) are used to study how metabolism varies in different physiological conditions. However, the great number of reactions involved in GEM makes it difficult to understand these variations. In order to have a more understandable tool, we developed a reduced metabolic model of central carbon and nitrogen metabolism, C2M2N with 77 reactions, 54 internal metabolites, and 3 compartments, taking into account the actual stoichiometry of the reactions, including the stoichiometric role of the cofactors and the irreversibility of some reactions. In order to model oxidative phosphorylation (OXPHOS) functioning, the proton gradient through the inner mitochondrial membrane is represented by two pseudometabolites DPH (∆pH) and DPSI (∆ψ). To illustrate the interest of such a reduced and quantitative model of metabolism in mammalian cells, we used flux balance analysis (FBA) to study all the possible fates of glutamine in metabolism. Our analysis shows that glutamine can supply carbon sources for cell energy production and can be used as carbon and nitrogen sources to synthesize essential metabolites. Finally, we studied the interplay between glucose and glutamine for the formation of cell biomass according to ammonia microenvironment. We then propose a quantitative analysis of the Warburg effect.

Quantitative measurement of pH dynamics in living cells using the mCherry‐TYG red fluorescent protein as a lifetime sensor

pH regulation plays a crucial role in mammalian and bacterial cell metabolism, protein function, apoptotic signaling, and a wide range of other physiological processes. Monitoring intracellular pH after environmental perturbations would further knowledge about cellular activities in both healthy and disease states in mammalian cells, as well as mechanism of infection and antibiotic resistance in bacterial cells. Non‐optical techniques such as x‐ray crystallography and NMR cannot resolve live‐cell dynamics, and many fluorescent dye or protein‐based sensors are susceptible to concentration and intensity dependence of the fluorophore, sample thickness, and photo‐bleaching, making lab‐to‐lab translation difficult without extensive calibration. Therefore, there remains a need for options to quantitatively measure pH dynamics in live specimens. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. The utilization of FRET pairs in fluorescence lifetime imaging microscopy (FLIM), has shown applicability of lifetime measurement for dynamic quantitation, however dynamic range can be limited. Here we present in vitro and live‐cell characterization of a red monomeric pH sensitive mCherry mutant mCherry‐TYG for robust measurement of live bacterial and mammalian pH in real‐time. We were successfully able to translate lifetime measurements with ease, showing pH calibration of both isolated protein and bacterial suspensions overlaying with a 2 ns dynamic range between pH 5.5 and 9.0. We further tested applicability in monitoring environmental perturbations by quantifying changes in pH as a function of energy source in e. coli. Additionally, we are currently exploring application for monitoring receptor trafficking, endocytosis and degradation in mammalian cells. Our results demonstrate the possibility of red shifted protein‐based lifetime sensors as a robust tool for pH calibration in live cells.Support or Funding InformationNIH/NINDS R21 NS092010, R21 NS106319NIH/NEI R21 EY026425This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microfluidic oxygen sensor system as a tool to monitor the metabolism of mammalian cells

We present a sensor system to monitor the uptake of oxygen by mammalian cells as a direct indicator for the metabolism under consideration of the requirements for the usage in biolabs. That includes reliable oxygen sensing as well as a small footprint of the setup without sophisticated external equipment, the usage of compatible materials and the suitability for a high degree of integration and automation. These requirements are fulfilled by a system that consists of a microfluidic chip with an integrated oxygen-sensitive phosphorescent film, heater and temperature sensor, external optical read-out and 3D-printed holders and housing. The chip with the closed microfluidic chamber is made by clean-room technologies out of silicon and glass. An excitation LED and a small and low-cost Raspberry Pi camera are used to measure the phosphorescent signal. With this system, the concentration of dissolved oxygen can be determined with an accuracy of ±0.8% (air) for oxygen concentrations between 0 and 26% (air) and at any temperature between 23 and 41 °C. To demonstrate the capabilities, the oxygen consumption rates of HaCaT-cells (human keratinocyte cell line) are determined at different temperatures.

Novel 3,6-Dihydroxypicolinic Acid Decarboxylase-Mediated Picolinic Acid Catabolism in Alcaligenes faecalis JQ135.

Picolinic acid (PA), a typical C-2-carboxylated pyridine derivative, is a metabolite of l-tryptophan and many other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize PA for growth. However, the precise mechanism of PA metabolism remains unknown. Alcaligenes faecalis strain JQ135 utilizes PA as its carbon and nitrogen source for growth. In this study, we screened a 6-hydroxypicolinic acid (6HPA) degradation-deficient mutant through random transposon mutagenesis. The mutant hydroxylated 6HPA into an intermediate, identified as 3,6-dihydroxypicolinic acid (3,6DHPA), with no further degradation. A novel decarboxylase, PicC, was identified to be responsible for the decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. Although, PicC belonged to the amidohydrolase 2 family, it shows low similarity (<45%) compared to other reported amidohydrolase 2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results demonstrated that picC was essential for PA degradation. The PicC was Zn2+-dependent nonoxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. The Km and kcat toward 3,6DHPA were observed to be 13.44 μM and 4.77 s-1, respectively. Site-directed mutagenesis showed that His163 and His216 were essential for PicC activity. This study provides new insights into the microbial metabolism of PA at molecular level.IMPORTANCE Picolinic acid is a natural toxic pyridine derived from l-tryptophan metabolism and other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize picolinic acid for their growth, and thus a microbial degradation pathway of picolinic acid has been proposed. Picolinic acid is converted into 6-hydroxypicolinic acid, 3,6-dihydroxypicolinic acid, and 2,5-dihydroxypyridine in turn. However, there was no physiological and genetic validation for this pathway. This study demonstrated that 3,6-dihydroxypicolinic acid was an intermediate in picolinic acid catabolism and further identified and characterized a novel amidohydrolase 2 family decarboxylase PicC. PicC was also shown to catalyze the decarboxylation of 3,6-dihydroxypicolinic acid into 2,5-dihydroxypyridine. This study provides a basis for understanding picolinic acid degradation and its underlying molecular mechanism.

A framework for tracer-based metabolism in mammalian cells by NMR

Metabolism changes extensively during the normal proliferation and differentiation of mammalian cells, and in cancer and inflammatory diseases. Since changes in the metabolic network reflect interactions between genetic, epigenetic and environmental changes, it is helpful to study the flow of label from isotopically labelled precursors into other metabolites rather than static metabolite levels. For this Nuclear Magnetic Resonance (NMR) spectroscopy is an attractive technique as it can quantify site-specific label incorporation. However, for applications using human cells and cell lines, the challenge is to optimize the process to maximize sensitivity and reproducibility. Here we present a new framework to analyze metabolism in mammalian cell lines and primary cells, covering the workflow from the preparation of cells to the acquisition and analysis of NMR spectra. We have applied this new approach in hematological and liver cancer cell lines and confirm the feasibility of tracer-based metabolism in primary liver cells.

New insights into GluT1 mechanics during glucose transfer

Glucose plays a crucial role in the mammalian cell metabolism. In the erythrocytes and endothelial cells of the blood-brain barrier, glucose uptake is mediated by the glucose transporter type 1 (GluT1). GluT1 deficiency or mutations cause severe physiological disorders. GluT1 is also an important target in cancer therapy as it is overexpressed in tumor cells. Previous studies have suggested that GluT1 mediates solute transfer through a cycle of conformational changes. However, the corresponding 3D structures adopted by the transporter during the transfer process remain elusive. In the present work, we first elucidate the whole conformational landscape of GluT1 in the absence of glucose, using long molecular dynamics simulations and show that the transitions can be accomplished through thermal fluctuations. Importantly, we highlight a strong coupling between intracellular and extracellular domains of the protein that contributes to the transmembrane helices reorientation during the transition. The conformations adopted during the simulations differ from the known 3D bacterial homologs structures resolved in similar states. In holo state simulations, we find that glucose transits along the pathway through significant rotational motions, while maintaining hydrogen bonds with the protein. These persistent motions affect side chains orientation, which impacts protein mechanics and allows glucose progression.

Tumor-suppressive function of SIRT4 in neuroblastoma through mitochondrial damage.

BackgroundSIRT4 is a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes located in the mitochondria, and is involved in regulating energy metabolism, stress response, and cellular lifespan in mammalian cells. However, its function in human neuroblastoma (NB) remains unexplored.MethodsExpression of SIRT4 in 158 pairs of human NB tumor tissues and adjacent normal tissues collected from March 2009 to October 2012 was analyzed by immunohistochemistry, Western blotting, and real-time fluorescence quantitative PCR. For in vitro study, SIRT4 was overexpressed in SH-SY5Y, SK-N-BE, and IMR-32 cells to study the effects of SIRT4 expression on proliferation, invasion, and migration of human NB cells and on mitochondrial function.ResultsSIRT4 gene expression in human NB tumor tissues was significantly lower than that in adjacent normal tissues (P<0.001). SIRT4 expression was lower in NB patients with higher International Neuroblastoma Staging System stage (P=0.018), with lymph node metastasis, than patients without lymph node metastasis (P<0.001). Survival times of NB patients with low expression of SIRT4 were significantly shorter than those of patients with high expression of SIRT4 (P=0.0036). Overexpression of SIRT4 significantly reduced the proliferation, invasion, and migration ability of NB cells as well as mitochondrial energy production, and caused SIRT1 upregulation and mitochondrial damage in NB cells.ConclusionSIRT4 exhibits a tumor suppressor function in human NB and inhibits mitochondrial metabolism and SIRT1 expression in tumor cells, thereby reducing the energy metabolism of tumor cells. These results suggest that SIRT4 may be a new therapeutic target for human NB.

Dynamics and functional interplay of histone lysine butyrylation, crotonylation, and acetylation in rice under starvation and submergence

BackgroundHistone lysine acylations by short-chain fatty acids are distinct from the widely studied histone lysine acetylation in chromatin, although both modifications are regulated by primary metabolism in mammalian cells. It remains unknown whether and how histone acylation and acetylation interact to regulate gene expression in plants that have distinct regulatory pathways of primary metabolism.ResultsWe identify 4 lysine butyrylation (Kbu) sites (H3K14, H4K12, H2BK42, and H2BK134) and 45 crotonylation (Kcr) sites on rice histones by mass spectrometry. Comparative analysis of genome-wide Kbu and Kcr and H3K9ac in combination with RNA sequencing reveals 25,306 genes marked by Kbu and Kcr in rice and more than 95% of H3K9ac-marked genes are marked by both. Kbu and Kcr are enriched at the 5′ region of expressed genes. In rice under starvation and submergence, Kbu and Kcr appear to be less dynamic and display changes in different sets of genes compared to H3K9ac. Furthermore, Kbu seems to preferentially poise gene activation by external stresses, rather than internal circadian rhythm which has been shown to be tightly associated with H3K9ac. In addition, we show that rice sirtuin histone deacetylase (SRT2) is involved in the removal of Kcr.ConclusionKbu, Kcr, and H3K9ac redundantly mark a large number of active genes but display different responses to external and internal signals. Thus, the proportion of rice histone lysine acetylation and acylation is dynamically regulated by environmental and metabolic cues, which may represent an epigenetic mechanism to fine-tune gene expression for plant adaptation.

RNF34 modulates the mitochondrial biogenesis and exercise capacity in muscle and lipid metabolism through ubiquitination of PGC-1 in Drosophila.

The transcriptional co-activator PGC-1α is a key regulator of mitochondrial function and muscle fiber specification in the skeletal muscle. The E3 ubiquitin ligase RNF34 ubiquitinates PGC-1α and negatively regulates mammalian brown fat cell metabolism. However, the functional importance of RNF34 in the skeletal muscle and its impact on energy metabolism remain unknown. The Drosophila PGC-1 homolog dPGC-1 and its mammalian counterparts have conserved functions in mitochondria and insulin signaling. Here, we showed that the Drosophila RNF34 (dRNF34) ubiquitinates the Drosophila PGC-1α (dPGC-1) and promotes its degradation in HEK293T cells by immunoprecipitation and western blot analysis. This allows us to use Drosophila as a powerful model system to study the physiological role of RNF34 in mitochondrial function and metabolism. In the in vivo studies, by separately expressing two independent UAS-dRNF34 RNAi transgenes driven by the muscle-specific 24B-Gal4 driver, we found that knockdown of dRNF34 specifically in muscle promotes mitochondrial biogenesis, improves negative geotaxis, extends climbing time to exhaustion in moderate aged flies and counteracts high-fat-diet-induced high triglyceride content. Furthermore, we showed that knockdown of dPGC-1 reversed the effects of the dRNF34 knockdown phenotypes described above. Our results reveal that dRNF34 plays an important role in regulating mitochondrial biogenesis in muscle and lipid metabolism through dPGC-1. Thus, inhibition of RNF34 activity provides a potential novel therapeutic strategy for the treatment of age-related muscle dysfunction.

A Cybernetic Approach to Modeling Lipid Metabolism in Mammalian Cells

The goal-oriented control policies of cybernetic models have been used to predict metabolic phenomena such as the behavior of gene knockout strains, complex substrate uptake patterns, and dynamic metabolic flux distributions. Cybernetic theory builds on the principle that metabolic regulation is driven towards attaining goals that correspond to an organism’s survival or displaying a specific phenotype in response to a stimulus. Here, we have modeled the prostaglandin (PG) metabolism in mouse bone marrow derived macrophage (BMDM) cells stimulated by Kdo2-Lipid A (KLA) and adenosine triphosphate (ATP), using cybernetic control variables. Prostaglandins are a well characterized set of inflammatory lipids derived from arachidonic acid. The transcriptomic and lipidomic data for prostaglandin biosynthesis and conversion were obtained from the LIPID MAPS database. The model parameters were estimated using a two-step hybrid optimization approach. A genetic algorithm was used to determine the population of near optimal parameter values, and a generalized constrained non-linear optimization employing a gradient search method was used to further refine the parameters. We validated our model by predicting an independent data set, the prostaglandin response of KLA primed ATP stimulated BMDM cells. We show that the cybernetic model captures the complex regulation of PG metabolism and provides a reliable description of PG formation.

Sequential Parameter Estimation for Mammalian Cell Model Based on In Silico Design of Experiments

Due to the complicated metabolism of mammalian cells, the corresponding dynamic mathematical models usually consist of large sets of differential and algebraic equations with a large number of parameters to be estimated. On the other hand, the measured data for estimating the model parameters are limited. Consequently, the parameter estimates may converge to a local minimum far from the optimal ones, especially when the initial guesses of the parameter values are poor. The methodology presented in this paper provides a systematic way for estimating parameters sequentially that generates better initial guesses for parameter estimation and improves the accuracy of the obtained metabolic model. The model parameters are first classified into four subsets of decreasing importance, based on the sensitivity of the model’s predictions on the parameters’ assumed values. The parameters in the most sensitive subset, typically a small fraction of the total, are estimated first. When estimating the remaining parameters with next most sensitive subset, the subsets of parameters with higher sensitivities are estimated again using their previously obtained optimal values as the initial guesses. The power of this sequential estimation approach is illustrated through a case study on the estimation of parameters in a dynamic model of CHO cell metabolism in fed-batch culture. We show that the sequential parameter estimation approach improves model accuracy and that using limited data to estimate low-sensitivity parameters can worsen model performance.

Understanding the interplay between amino acid and lipid metabolism in tumor growth

Metabolism is central to virtually all cellular functions and contributes to a range of diseases. A quantitative understanding of how biochemical pathways are dysregulated in the context of diseases such as cancer and metabolic syndrome is necessary to identify new therapeutic targets. To this end we apply stable isotope tracers, mass spectrometry, and metabolic flux analysis (MFA) to study metabolism in mammalian cells, animal models, and human patients. Using these approaches we have characterized how proliferating and differentiated cells regulate flux of glucose and amino acids into lipid biosynthetic pathways. We are particularly interested in understanding how amino acid and lipid metabolism are coordinated to meet these needs. Transport of pyruvate into mitochondria is a critical step in these processes, and activity of the mitochondrial pyruvate carrier (MPC) strongly influences cancer growth in vivo and as tumor spheroids. Using metabolomics and lipidomics approaches we have identified mechanisms through which MPC flux and amino acid availability impact sphingolipid metabolism. Modulation of these pathways influences tumor growth, providing mechanistic insights into how amino acid metabolism can influence lipids and mitochondrial function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Redox-dependent axial ligand replacement and its functional significance in heme-bound iron regulatory proteins

Iron regulatory proteins (IRPs), regulators of iron metabolism in mammalian cells, control the translation of proteins involved in iron uptake, storage and utilization by binding to specific iron-responsive element (IRE) sequences of mRNAs. Two homologs of IRPs (IRP1 and IRP2) have a typical heme regulatory motif (HRM), a consensus sequence found in “heme-regulated proteins”. However, specific heme binding to HRM has been reported only for IRP2, which is essential for oxidative modification and loss of binding to target mRNAs. In this paper, we confirmed that IRP1 also specifically binds two molar equivalents of heme, and found that the absorption and resonance Raman spectra of heme-bound IRP1 were quite similar to those of heme-bound IRP2. This shows that the heme environmental structures in IRP1 are close to those of proteins using heme as a regulatory molecule. Pulse radiolysis experiments, however, clearly revealed an axial ligand exchange from Cys to His immediately after the reduction of the heme iron to form a 5-coordinate His-ligated heme in heme-bound IRP2, whereas the 5-coordinate His-ligated heme was not observed after the reduction of heme-bound IRP1. Considering that the oxidative modification is only observed in heme-bound IRP2, but not IRP1, probably owing to the structural flexibility of IRP2, we propose that the transient 5-coordinate His-ligated heme is a prerequisite for oxidative modification of heme-bound IRP2, which functionally differentiates heme binding of IRP2 from that of IRP1.