Rapid probing of glucose influx into cancer cell metabolism: using adjuvant and a pH-dependent collection of central metabolites to improve in-cell D-DNP NMR.

Changes to metabolism are a hallmark of many diseases. Disease metabolism under physiological conditions can be probed in real time with in-cell NMR assays. Here, we pursued a systematic approach towards improved in-cell NMR assays. Unambiguous identifications of metabolites and of intracellular pH are afforded by a comprehensive, downloadable collection of spectral data for central carbon metabolites in the physiological pH range (4.0-8.0). Chemical shifts of glycolytic intermediates provide unique pH dependent patterns akin to a barcode. Using hyperpolarized 13C1 enriched glucose as the probe molecule of central metabolism in cancer, we find that early glycolytic intermediates are detectable in PC-3 prostate cancer cell lines, concurrently yielding intracellular pH. Using non-enriched and non-enhanced pyruvate as an adjuvant, reactions of the pentose phosphate pathway become additionally detectable, without significant changes to the barriers in upper glycolysis and to intracellular pH. The scope of tracers for in-cell observations can thus be improved by the presence of adjuvants, showing that a recently proposed effect of pyruvate in the tumor environment is paralleled by a rerouting of cancer cell metabolism towards producing building blocks for proliferation. Overall, the combined use of reference data for compound identification, site specific labelling for reducing overlap, and use of adjuvant afford increasingly detailed insight into disease metabolism.

Conversion of Similar Xenochemicals to Dissimilar Products: Exploiting Competing Reactions in Whole-Cell Catalysis.

Many enzymes have latent activities that can be used in the conversion of non-natural reactants for novel organic conversions. A classic example is the conversion of benzaldehyde to a phenylacetyl carbinol, a precursor for ephedrine manufacture. It is often tacitly assumed that purified enzymes are more promising catalysts than whole cells, despite the lower cost and easier maintenance of the latter. Competing substrates inside the cell have been known to elicit currently hard-to-predict selectivities that are not easily measured inside the living cell. We employ NMR spectroscopic assays to rationally combine isomers for selective reactions in commercial S. cerevisiae. This approach uses internal competition between alternative pathways of aldehyde clearance in yeast, leading to altered selectivities compared to catalysis with the purified enzyme. In this manner, 4-fluorobenzyl alcohol and 2-fluorophenylacetyl carbinol can be formed with selectivities in the order of 90%. Modification of the cellular redox state can be used to tune product composition further. Hyperpolarized NMR shows that the cellular reaction and pathway usage are affected by the xenochemical. Overall, we find that the rational construction of ternary or more complex substrate mixtures can be used for in-cell NMR spectroscopy to optimize the upgrading of similar xenochemicals to dissimilar products with cheap whole-cell catalysts.

Formate Metabolism in Shigella flexneri and Its Effect on HeLa Cells at Different Stages during the Infectious Process.

Shigellosis caused by Shigella is one of the most important foodborne illnesses in global health, but little is known about the metabolic cross talk between this bacterial pathogen and its host cells during the different stages of the infection process. A detailed understanding of the metabolism can potentially lead to new drug targets remedying the pressing problem of antibiotic resistance. Here, we use stable isotope-resolved metabolomics as an unbiased and fast method to investigate how Shigella metabolizes 13C-glucose in three different environments: inside the host cells, adhering to the host cells, and alone in suspension. We find that especially formate metabolism by bacteria is sensitive to these different environments. The role of formate in pathogen metabolism is sparsely described in the literature compared to the roles of acetate and butyrate. However, its metabolic pathway is regarded as a potential drug target due to its production in microorganisms and its absence in humans. Our study provides new knowledge about the regulatory effect of formate. Bacterial metabolism of formate is pH dependent when studied alone in culture medium, whereas this effect is less pronounced when the bacteria adhere to the host cells. Once the bacteria are inside the host cells, we find that formate accumulation is reduced. Formate also affects the host cells resulting in a reduced infection rate. This was correlated to an increased immune response. Thus, intriguingly formate plays a double role in pathogenesis by increasing the virulence of Shigella and at the same time stimulating the immune response of the host. IMPORTANCE Bacterial infection is a pressing societal concern due to development of resistance toward known antibiotics. Central carbon metabolism has been suggested as a potential new target for drug development, but metabolic changes upon infection remain incompletely understood. Here, we used a cellular infection model to study how the bacterial pathogen Shigella adapts its metabolism depending on the environment starting from the extracellular medium until Shigella successfully invaded and proliferated inside host cells. The mixed-acid fermentation of Shigella was the major metabolic pathway during the infectious process, and the glucose-derived metabolite formate surprisingly played a divergent role in the pathogen and in the host cell. Our data show reduced infection rate when both host cells and bacteria were treated with formate, which correlated with an upregulated immune response in the host cells. The formate metabolism in Shigella thus potentially provides a route toward alternative treatment strategies for Shigella prevention.

In-Cell NMR Approach for Real-Time Exploration of Pathway Versatility: Substrate Mixtures in Nonengineered Yeast.

The central carbon metabolism of microbes will likely be used in future sustainable bioproduction. A sufficiently deep understanding of central metabolism would advance the control of activity and selectivity in whole-cell catalysis. Opposite to the more obvious effects of adding catalysts through genetic engineering, the modulation of cellular chemistry through effectors and substrate mixtures remains less clear. NMR spectroscopy is uniquely suited for in-cell tracking to advance mechanistic insight and to optimize pathway usage. Using a comprehensive and self-consistent library of chemical shifts, hyperpolarized NMR, and conventional NMR, we probe the versatility of cellular pathways to changes in substrate composition. Conditions for glucose influx into a minor pathway to an industrial precursor (2,3-butanediol) can thus be designed. Changes to intracellular pH can be followed concurrently, while mechanistic details for the minor pathway can be derived using an intermediate-trapping strategy. Overflow at the pyruvate level can be induced in nonengineered yeast with suitably mixed carbon sources (here glucose with auxiliary pyruvate), thus increasing glucose conversion to 2,3-butanediol by more than 600-fold. Such versatility suggests that a reassessment of canonical metabolism may be warranted using in-cell spectroscopy.

Classification and biomarker identification of prostate tissue from TRAMP mice with hyperpolarized 13C-SIRA.

Hyperpolarized 13C isotope resolved spectroscopy boosts NMR signal intensity, which improves signal detection and allows metabolic fluxes to be analyzed. Such hyperpolarized flux data may offer new approaches to tissue classification and biomarker identification that could be translated in vivo. Here we used hyperpolarized stable isotope resolved analysis (SIRA) to measure metabolite specific 13C isotopic enrichments in the central carbon metabolism of mouse prostate. Prostate and tumor tissue samples were acquired from transgenic adenocarcinomas of the mouse prostate (TRAMP) mice. Before euthanasia, mice were injected with [U-13C]glucose intraperitoneally (i.p.). Polar metabolite extracts were prepared, and hyperpolarized 1D-13C NMR spectra were obtained from normal prostate (n = 19) and cancer tissue (n = 19) samples. Binary classification and feature analysis was performed to make a separation model and to investigate differences between samples originating from normal and cancerous prostate tissue, respectively. Hyperpolarized experiments were carried out according to a standardized protocol, which showed a high repeatability (CV = 15%) and an average linewidth in the 1D-13C NMR spectra of 2 ± 0.5 Hz. The resolution of the hyperpolarized 1D-13C spectra was high with little signal overlap in the carbonyl region and metabolite identification was easily accomplished. A discrimination with 95% success rate could be made between samples originating from TRAMP mice prostate and tumor tissue based on isotopomers from uniquely identified metabolites. Hyperpolarized 13C-SIRA allowed detailed metabolic information to be obtained from tissue specimens. The positional information of 13C isotopic enrichments lead to easily interpreted features responsible for high predictive classification of tissue types. This analytical approach has matured, and the robust experimental protocols currently available allow systematic tracking of metabolite flux ex vivo.

Enhanced 13C NMR detects extended reaction networks in living cells.

Insights into intracellular chemistry have remained sparse, but would be impactful for the advancement of biomedicine and bioproduction. A suitable 13C NMR approach provides improvements in sensitivity that make extended reaction networks and assay time windows, previously inaccessible cell densities and relative flux measurements accessible in living cells.

Stable isotope resolved metabolomics classification of prostate cancer cells using hyperpolarized NMR data.

Metabolic fingerprinting is a strong tool for characterization of biological phenotypes. Classification with machine learning is a critical component in the discrimination of molecular determinants. Cellular activity can be traced using stable isotope labelling of metabolites from which information on cellular pathways may be obtained. Nuclear magnetic resonance (NMR) spectroscopy is, due to its ability to trace labelling in specific atom positions, a method of choice for such metabolic activity measurements. In this study, we used hyperpolarization in the form of dissolution Dynamic Nuclear Polarization (dDNP) NMR to measure signal enhanced isotope labelled metabolites reporting on pathway activity from four different prostate cancer cell lines. The spectra have a high signal-to-noise, with less than 30 signals reporting on 10 metabolic reactions. This allows easy extraction and straightforward interpretation of spectral data. Four metabolite signals selected using a Random Forest algorithm allowed a classification with Support Vector Machines between aggressive and indolent cancer cells with 96.9% accuracy, -corresponding to 31 out of 32 samples. This demonstrates that the information contained in the few features measured with dDNP NMR, is sufficient and robust for performing binary classification based on the metabolic activity of cultured prostate cancer cells.

Catalytic cycle of carbohydrate dehydration by Lewis acids: structures and rates from synergism of conventional and DNP NMR.

Lewis acids play key roles in many chemical reactions. Structural and functional (kinetic) detail in Lewis acid catalysed fructose conversion are derived herein by the combined use of conventional and dissolution dynamic nuclear polarization (D-DNP) NMR. Structural information obtained with D-DNP NMR was used to identify conditions that stabilize an elusive initial intermediate and to determine its chemical structure. Carbohydrate dehydration through this intermediate had been predicted computationally. Complementary kinetic NMR assays yielded rate constants spanning three orders of magnitude for the three biggest energy barriers in the catalytic cycle.

Discovery and Exploration of the Efficient Acyclic Dehydration of Hexoses in Dimethyl Sulfoxide/Water.

Current gaps in the development of sustainable processes include a lack of strategies to systematically identify and optimize the formation of new products. The dehydration of hexoses to 5-hydroxymethylfurfural (HMF) is a particularly widely studied process. In an attempt to identify a new high-selectivity conversion of glucose, quantitative NMR spectroscopy is used to screen conditions that have been reported to yield high conversions of glucose but low formation of HMF. In this manner, an olefinic six-carbon byproduct is identified. By adding water, selectivity for the compound was nearly tripled relative to previous reports. The detection of high-yielding side reactions in the formation of HMF is remarkable, considering how extensively HMF formation has been studied. High selectivity for the acyclic pathway allows hitherto unobserved intermediates in this pathway to be identified by using in situ NMR spectroscopy. An additional, presumably cyclic, pathway contributes to HMF formation.

Difference between Extra- and Intracellular T1 Values of Carboxylic Acids Affects the Quantitative Analysis of Cellular Kinetics by Hyperpolarized NMR.

Incomplete knowledge of the longitudinal relaxation time constant (T1 ) leads to incorrect assumptions in quantitative kinetic models of cellular systems, studied by hyperpolarized real-time NMR. Using an assay that measures the intracellular signal of small carboxylic acids in living cells, the intracellular T1 of the carboxylic acid moiety of acetate, keto-isocaproate, pyruvate, and butyrate was determined. The intracellular T1 is shown to be up to four-fold shorter than the extracellular T1 . Such a large difference in T1 values between the inside and the outside of the cell has significant influence on the quantification of intracellular metabolic activity. It is expected that the significantly shorter T1 value of the carboxylic moieties inside cells is a result of macromolecular crowding. An artificial cytosol has been prepared and applied to predict the T1 of other carboxylic acids. We demonstrate the value of this prediction tool.

Hyperpolarised organic phosphates as NMR reporters of compartmental pH.

Organic phosphate metabolites contain functional groups with pKa values near the physiologic pH range, yielding pH-dependent (13)C chemical shift changes of adjacent quaternary carbon sites. When formed in defined cellular compartments from exogenous hyperpolarised (13)C substrates, metabolites can thus yield localised pH values and correlations of organelle pH and catalytic activity.

Spectroscopic approaches to resolving ambiguities of hyper-polarized NMR signals from different reaction cascades.

The influx of exogenous substrates into cellular reaction cascades on the seconds time scale is directly observable by NMR spectroscopy when using nuclear spin polarization enhancement. Conventional NMR assignment spectra for the identification of reaction intermediates are not applicable in these experiments due to the non-equilibrium nature of the nuclear spin polarization enhancement. We show that ambiguities in the intracellular identification of transient reaction intermediates can be resolved by experimental schemes using site-specific isotope labelling, optimised referencing and response to external perturbations.

Probing treatment response of glutaminolytic prostate cancer cells to natural drugs with hyperpolarized [5-(13) C]glutamine.

PURPOSE: The correlation between glutamine metabolism and oncogene expression in cancers has led to a renewed interest in the role of glutamine in cancer cell survival. Hyperpolarized [5-(13) C]glutamine is evaluated as a potential biomarker for noninvasive metabolic measurements of drug response in prostate cancer cells. METHODS: Hyperpolarized [5-(13) C]glutamine is used to measure glutamine metabolism in two prostate cancer cell lines (PC3 and DU145) before and after treatment with the two natural anticancer drugs resveratrol and sulforaphane. An invasive biochemical assay simulating the hyperpolarized experiment is used to independently quantify glutamine metabolism. RESULTS: Glutamine metabolism is found to be 4 times higher in the more glutaminolytic DU145 cells compared with PC3 cells under proliferating growth conditions by using hyperpolarized [5-(13) C]glutamine as a noninvasive probe. A significant decrease in glutamine metabolism occurs upon apoptotic response to treatment with resveratrol and sulforaphane. CONCLUSION: Hyperpolarized NMR using [5-(13) C]glutamine as a probe permits the noninvasive observation of glutaminolysis in different cell lines and under different treatment conditions. Hyperpolarized [5-(13) C]glutamine metabolism thus is a promising biomarker for the noninvasive detection of tumor response to treatment, as it directly monitors one of the hallmarks in cancer metabolism - glutaminolysis - in living cells.

Structural basis and dynamics of multidrug recognition in a minimal bacterial multidrug resistance system.

TipA is a transcriptional regulator found in diverse bacteria. It constitutes a minimal autoregulated multidrug resistance system against numerous thiopeptide antibiotics. Here we report the structures of its drug-binding domain TipAS in complexes with promothiocin A and nosiheptide, and a model of the thiostrepton complex. Drug binding induces a large transition from a partially unfolded to a globin-like structure. The structures rationalize the mechanism of promiscuous, yet specific, drug recognition: (i) a four-ring motif present in all known TipA-inducing antibiotics is recognized specifically by conserved TipAS amino acids; and (ii) the variable part of the antibiotic is accommodated within a flexible cleft that rigidifies upon drug binding. Remarkably, the identified four-ring motif is also the major interacting part of the antibiotic with the ribosome. Hence the TipA multidrug resistance mechanism is directed against the same chemical motif that inhibits protein synthesis. The observed identity of chemical motifs responsible for antibiotic function and resistance may be a general principle and could help to better define new leads for antibiotics.

Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes.

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.

Sequence-structure and structure-function analysis in cysteine-rich domains forming the ultrastable nematocyst wall.

The nematocyst wall of cnidarians is a unique biomaterial that withstands extreme osmotic pressures, allowing an ultrafast discharge of the nematocyst capsules. Assembly of the highly robust nematocyst wall is achieved by covalent linkage of cysteine-rich domains (CRDs) from two main protein components, minicollagens and nematocyst outer wall antigen (NOWA). The bipolar minicollagens have different disulfide patterns and topologies in their N and C-terminal CRDs. The functional significance of this polarity has been elusive. Here, we show by NMR structural analysis that all representative cysteine-rich domains of NOWA are structurally related to N-terminal minicollagen domains. Natural sequence insertions in NOWA CRDs have very little effect on the tightly knit domain structures, nor do they preclude the efficient folding to a single native conformation. The different folds in NOWA CRDs and the atypical C-terminal minicollagen domain on the other hand can be directly related to different conformational preferences in the reduced states. Ultrastructural analysis in conjunction with aggregation studies argues for an association between the similar NOWA and N-terminal minicollagen domains in early stages of the nematocyst wall assembly, which is followed by the controlled association between the unusual structures of C-terminal minicollagen domains.

Improvement of hydrogen bond geometry in protein NMR structures by residual dipolar couplings--an assessment of the interrelation of NMR restraints.

We have examined how the hydrogen bond geometry in three different proteins is affected when structural restraints based on measurements of residual dipolar couplings are included in the structure calculations. The study shows, that including restraints based solely on (1)H(N)-(15)N residual dipolar couplings has pronounced impact on the backbone rmsd and Ramachandran plot but does not improve the hydrogen bond geometry. In the case of chymotrypsin inhibitor 2 the addition of (13)CO-(13)C(alpha) and (15)N-(13)CO one bond dipolar couplings as restraints in the structure calculations improved the hydrogen bond geometry to a quality comparable to that obtained in the 1.8 A resolution X-ray structure of this protein. A systematic restraint study was performed, in which four types of restraints, residual dipolar couplings, hydrogen bonds, TALOS angles and NOEs, were allowed in two states. This study revealed the importance of using several types of residual dipolar couplings to get good hydrogen bond geometry. The study also showed that using a small set of NOEs derived only from the amide protons, together with a full set of residual dipolar couplings resulted in structures of very high quality. When reducing the NOE set, it is mainly the side-chain to side-chain NOEs that are removed. Despite of this the effect on the side-chain packing is very small when a reduced NOE set is used, which implies that the over all fold of a protein structure is mainly determined by correct folding of the backbone.