Combined In-Cell NMR and Simulation Approach to Probe Redox-Dependent Pathway Control.

Dynamic response of intracellular reaction cascades to changing environments is a hallmark of living systems. As metabolism is complex, mechanistic models have gained popularity for describing the dynamic response of cellular metabolism and for identifying target genes for engineering. At the same time, the detailed tracking of transient metabolism in living cells on the subminute time scale has become amenable using dynamic nuclear polarization-enhanced 13C NMR. Here, we suggest an approach combining in-cell NMR spectroscopy with perturbation experiments and modeling to obtain evidence that the bottlenecks of yeast glycolysis depend on intracellular redox state. In pre-steady-state glycolysis, pathway bottlenecks shift from downstream to upstream reactions within a few seconds, consistent with a rapid decline in the NAD+/NADH ratio. Simulations using mechanistic models reproduce the experimentally observed response and help identify unforeseen biochemical events. Remaining inaccuracies in the computational models can be identified experimentally. The combined use of rapid injection NMR spectroscopy and in silico simulations provides a promising method for characterizing cellular reactions with increasing mechanistic detail.

Stable Isotope-Resolved Analysis with Quantitative Dissolution Dynamic Nuclear Polarization.

Metabolite profiles and their isotopomer distributions can be studied noninvasively in complex mixtures with NMR. The advent of dissolution Dynamic Nuclear Polarization (dDNP) and isotope enrichment add sensitivity and resolution to such metabolic studies. Metabolic pathways and networks can be mapped and quantified if protocols that control and exploit the ex situ signal enhancement are created. We present a sample preparation method, including cell incubation, extraction and signal enhancement, to obtain reproducible and quantitative dDNP (qdDNP) NMR-based stable isotope-resolved analysis. We further illustrate how qdDNP was applied to gain metabolic insights into the phenotype of aggressive cancer cells.

Hyperpolarized [1,3-13C2 ]ethyl acetoacetate is a novel diagnostic metabolic marker of liver cancer.

An increased prevalence of liver diseases such as hepatitis C and nonalcoholic fatty liver results in an augmented incidence of the most common form of liver cancer, hepatocellular carcinoma (HCC). HCC is most often found in the cirrhotic liver and it can therefore be challenging to rely on anatomical information alone when diagnosing HCC. Valuable information on specific cellular metabolism can be obtained with high sensitivity thanks to an emerging magnetic resonance (MR) technique that uses 13C labeled hyperpolarized molecules. Our interest was to explore potential new high contrast metabolic markers of HCC using hyperpolarized 13C-MR. This work led to the identification of a class of substrates, low molecular weight ethyl-esters, which showed high specificity for carboxyl esterases and proved in many cases to possess good properties for signal enhancement. In particular, hyperpolarized [1,3-13C2 ]ethyl acetoacetate (EAA) was shown to provide a metabolic fingerprint of HCC. Using this substrate a liver cancer implanted in rats was diagnosed as a consequence of an ∼4 times higher metabolic substrate-to-product ratio than in the surrounding healthy tissue, (p=0.009). Unregulated cellular uptake as well as cosubstrate independent enzymatic conversion of EAA, made this substrate highly useful as a hyperpolarized 13C-MR marker. This could be appreciated by the signal-to-noise (SNR) obtained from EAA, which was comparable to the SNR reported in a literature liver cancer study with state-of-the-art hyperpolarized substrate, [1-13C]pyruvate. Also, the contrast-to-noise (CNR) in the EAA based metabolic ratio images was significantly improved compared with the CNR in equivalent images reported using [1-13C]pyruvate.

Inhibition of beta cell growth and function by bone morphogenetic proteins.

AIMS/HYPOTHESIS: Impairment of beta cell mass and function is evident in both type 1 and type 2 diabetes. In healthy physiological conditions pancreatic beta cells adapt to the body's increasing insulin requirements by proliferation and improved function. We hypothesised that during the development of diabetes, there is an increase in the expression of inhibitory factors that prevent the beta cells from adapting to the increased need for insulin. We evaluated the effects of bone morphogenetic protein (BMP) 2 and -4 on beta cells. METHODS: The effects of BMP2 and -4 on beta cell proliferation, apoptosis, gene expression and insulin release were studied in isolated islets of Langerhans from rats, mice and humans. The expression of BMPs was analysed by immunocytochemistry and real-time PCR. The role of endogenous BMP was investigated using a soluble and neutralising form of the BMP receptor 1A. RESULTS: BMP2 and -4 were found to inhibit basal as well as growth factor-stimulated proliferation of primary beta cells from rats and mice. Bmp2 and Bmp4 mRNA and protein were expressed in islets and regulated by inflammatory cytokines. Neutralisation of endogenous BMP activity resulted in enhanced proliferation of rodent beta cells. The expression of Id mRNAs was induced by BMP4 in rat and human islets. Finally, glucose-induced insulin secretion was significantly impaired in rodent and human islets pre-treated with BMP4, and inhibition of BMP activity resulted in enhanced insulin release. CONCLUSIONS/INTERPRETATION: These data show that BMP2 and -4 exert inhibitory actions on beta cells in vitro and suggest that BMPs exert regulatory roles of beta cell growth and function.

WhiB7, an Fe-S-dependent transcription factor that activates species-specific repertoires of drug resistance determinants in actinobacteria.

WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.

Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.

As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H(13)CO(3)(-)) and (13)CO(2) following intravenous injection of hyperpolarized H(13)CO(3)(-). The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.

Hyperpolarized NMR probes for biological assays.

During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized) molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

Real-time DNP NMR observations of acetic acid uptake, intracellular acidification, and of consequences for glycolysis and alcoholic fermentation in yeast.

Uptake and upshot in vivo: Straightforward methods that permit the real-time observation of organic acid influx, intracellular acidification, and concomitant effects on cellular-reaction networks are crucial for improved bioprocess monitoring and control. Herein, dynamic nuclear polarization (DNP) NMR is used to observe acetate influx, ensuing intracellular acidification and the metabolic consequences on alcoholic fermentation and glycolysis in living cells.

Sulfite action in glycolytic inhibition: in vivo real-time observation by hyperpolarized (13)C NMR spectroscopy.

Detecting the molecular targets of xenobiotic substances in vivo poses a considerable analytical challenge. Here, we describe the use of an NMR-based tracer methodology for the instantaneous in vivo observation of sulfur(IV) action on cellular metabolism. Specifically, we find that glycolytic flux is directed towards sulfite adducts of dihydroxyacetone phosphate and pyruvate as off-pathway intermediates that obstruct glycolytic flux. In particular, the pyruvate-sulfite association hinders the formation of downstream metabolites. The apparent in vivo association constant of pyruvate and sulfite agrees with the apparent inhibition constant of CO(2) formation, thus supporting the importance of pyruvate interception in disturbing central metabolism and inhibiting NAD regeneration.

Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors.

Dynamic nuclear polarization of (13)C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-(13)C(2)]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-(13)C(2)]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized (13)C-labeled malate from [1,4-(13)C(2)]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically.

Quantitative dynamic nuclear polarization-NMR on blood plasma for assays of drug metabolism.

Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring.

Tissue-specific short chain fatty acid metabolism and slow metabolic recovery after ischemia from hyperpolarized NMR in vivo.

Mechanistic details of mammalian metabolism in vivo and dynamic metabolic changes in intact organisms are difficult to monitor because of the lack of spatial, chemical, or temporal resolution when applying traditional analytical tools. These limitations can be addressed by sensitivity enhancement technology for fast in vivo NMR assays of enzymatic fluxes in tissues of interest. We apply this methodology to characterize organ-specific short chain fatty acid metabolism and the changes of carnitine and coenzyme A pools in ischemia reperfusion. This is achieved by assaying acetyl-CoA synthetase and acetyl-carnitine transferase catalyzed transformations in vivo. The fast and predominant flux of acetate and propionate signal into acyl-carnitine pools shows the efficient buffering of free CoA levels. Sizeable acetyl-carnitine formation from exogenous acetate is even found in liver, where acetyl-CoA synthetase and acetyl-carnitine transferase activities have been assumed sequestered in different compartments. In vivo assays of altered acetate metabolism were applied to characterize pathological changes of acetate metabolism upon ischemia. Coenzyme pools in ischemic skeletal muscle are reduced in vivo even 1 h after disturbing muscle perfusion. Impaired mitochondrial metabolism and slow restoration of free CoA are corroborated by assays employing fumarate to show persistently reduced tricarboxylic acid (TCA) cycle activity upon ischemia. In the same animal model, anaerobic metabolism of pyruvate and tissue perfusion normalize faster than mitochondrial bioenergetics.

Imaging of branched chain amino acid metabolism in tumors with hyperpolarized 13C ketoisocaproate.

Powerful analytical tools are vital for characterizing the complex molecular changes underlying oncogenesis and cancer treatment. This is particularly true, if information is to be collected in vivo by noninvasive approaches. In the recent past, hyperpolarized (13)C magnetic resonance (MR) spectroscopy has been employed to quickly collect detailed spectral information on the chemical fate of tracer molecules in different tissues at high sensitivity. Here, we report a preclinical study showing that alpha-ketoisocaproic acid (KIC) can be used to assess molecular signatures of tumors with hyperpolarized MR spectroscopy. KIC is metabolized to leucine by the enzyme branched chain amino acid transferase (BCAT), which is found upregulated in some tumors. BCAT is a putative marker for metastasis and a target of the proto-oncogene c-myc. Very different fluxes through the BCAT-catalyzed reaction can be detected for murine lymphoma (EL4) and rat mammary adenocarcinoma (R3230AC) tumors in vivo. EL4 tumors show a more than 7-fold higher hyperpolarized (13)C leucine signal relative to the surrounding healthy tissue. In R3230AC tumor on the other hand branched chain amino acid metabolism is not enhanced relative to surrounding tissues. The distinct molecular signatures of branched chain amino acid metabolism in EL4 and R3230AC tumors correlate well with ex vivo assays of BCAT activity.

Continuous molecular evolution of protein-domain structures by single amino acid changes.

Protein structures cluster into families of folds that can result from extremely different amino acid sequences [1]. Because the enormous amount of genetic information generates a limited number of protein folds [2], a particular domain structure often assumes numerous functions. How new protein structures and new functions evolve under these limitations remains elusive. Molecular evolution may be driven by the ability of biomacromolecules to adopt multiple conformations as a bridge between different folds [3-6]. This could allow proteins to explore new structures and new tasks while part of the structural ensemble retains the initial conformation and function as a safeguard [7]. Here we show that a global structural switch can arise from single amino acid changes in cysteine-rich domains (CRD) of cnidarian nematocyst proteins. The ability of these CRDs to form two structures with different disulfide patterns from an identical cysteine pattern is distinctive [8]. By applying a structure-based mutagenesis approach, we demonstrate that a cysteine-rich domain can interconvert between two natively occurring domain structures via a bridge state containing both structures. Comparing cnidarian CRD sequences leads us to believe that the mutations we introduced to stabilize each structure reflect the birth of new protein folds in evolution.

Pancreatic ß-cells respond to fuel pressure with an early metabolic switch.

Pancreatic ß-cells become irreversibly damaged by long-term exposure to excessive glucose concentrations and lose their ability to carry out glucose stimulated insulin secretion (GSIS) upon damage. The ß-cells are not able to control glucose uptake and they are therefore left vulnerable for endogenous toxicity from metabolites produced in excess amounts upon increased glucose availability. In order to handle excess fuel, the ß-cells possess specific metabolic pathways, but little is known about these pathways. We present a study of ß-cell metabolism under increased fuel pressure using a stable isotope resolved NMR approach to investigate early metabolic events leading up to ß-cell dysfunction. The approach is based on a recently described combination of 13C metabolomics combined with signal enhanced NMR via dissolution dynamic nuclear polarization (dDNP). Glucose-responsive INS-1 ß-cells were incubated with increasing concentrations of [U-13C] glucose under conditions where GSIS was not affected (2-8 h). We find that pyruvate and DHAP were the metabolites that responded most strongly to increasing fuel pressure. The two major divergence pathways for fuel excess, the glycerolipid/fatty acid metabolism and the polyol pathway, were found not only to operate at unchanged rate but also with similar quantity.

Targeted Metabolomics with Quantitative Dissolution Dynamic Nuclear Polarization.

Metabolite profiles and their isotopomer distributions can be studied noninvasively in complex mixtures with NMR. The advent of hyperpolarized 13C-NMR using quantitative dissolution Dynamic Nuclear Polarization (qdDNP) and isotope enrichment add sensitivity to such metabolic studies, enabling mapping and quantification of metabolic pathways and networks. Here we describe a sample preparation method, including cell incubation, extraction, and signal enhancement, for reproducible and quantitative analysis of hyperpolarized 13C-NMR metabolite spectra. We further illustrate how qdDNP can be applied to gain metabolic insights into living cells.

Real-time detection of central carbon metabolism in living Escherichia coli and its response to perturbations.

The direct tracking of cellular reactions in vivo has been facilitated with recent technologies that strongly enhance NMR signals in substrates of interest. This methodology can be used to assay intracellular reactions that occur within seconds to few minutes, as the NMR signal enhancement typically fades on this time scale. Here, we show that the enhancement of (13)C nuclear spin polarization in deuterated glucose allows to directly follow the flux of glucose signal through rather extended reaction networks of central carbon metabolism in living Escherichia coli. Alterations in central carbon metabolism depending on the growth phase or upon chemical perturbations are visualized with minimal data processing by instantaneous observation of cellular reactions.