One touch is all it takes: the supramolecular interaction between ubiquitin and lanthanide complexes revisited by paramagnetic NMR and molecular dynamics.

The supramolecular interaction between lanthanide complexes and proteins is at the heart of numerous chemical and biological studies. Some of these complexes have demonstrated remarkable interaction properties with proteins or peptides in solution and in the crystalline state. Here we have used the paramagnetism of lanthanide ions to characterize the affinity of two lanthanide complexes for ubiquitin. As the interaction process is dynamic, the acquired NMR data only reflect the time average of the different steps. We have used molecular dynamics (MD) simulations to get a deeper insight into the detailed interaction scenario at the microsecond scale. This NMR/MD approach enabled us to establish that the tris-dipicolinate complex interacts specifically with arginines and lysines, while the crystallophore explores the protein surface through weak interactions with carboxylates. These observations shed new light on the dynamic interaction properties of these complexes, which will ultimately enable us to propose a crystallization mechanism.

NMR metabolomics study of chronic low-dose exposure to a cocktail of persistent organic pollutants.

Nowadays, exposure to endocrine-disrupting chemicals (EDCs), including persistent organic pollutants (POPs), is one of the most critical threats to public health. EDCs are chemicals that mimic, block, or interfere with hormones in the body's endocrine system and have been associated with a wide range of health issues. This innovative, untargeted metabolomics study investigates chronic low-dose internal exposure to a cocktail of POPs on multiple tissues that are known to accumulate these lipophilic compounds. Interestingly, the metabolic response differs among selected tissues/organs in mice. In the liver, we observed a dynamic effect according to the exposure time and the doses of POPs. In the brain tissue, the situation is the opposite, leading to the conclusion that the presence of POPs immediately gives a saturated effect that is independent of the dose and the duration of exposure studied. By contrast, for the adipose tissues, nearly no effect is observed. This metabolic profiling leads to a holistic and dynamic overview of the main metabolic pathways impacted in lipophilic tissues by a cocktail of POPs.

Present and future of pure shift NMR in metabolomics.

NMR is one of the most powerful techniques for the analysis of biological samples in the field of metabolomics. However, the high complexity of fluids, tissues, or other biological materials taken from living organisms is still a challenge for state-of-the-art pulse sequences, thereby limiting the detection, the identification, and the quantification of metabolites. In this context, the resolution enhancement provided by broadband homonuclear decoupling methods, which allows for simplifying 1 H multiplet patterns into singlets, has placed this so-called pure shift technique as a promising approach to perform metabolic profiling with unparalleled level of detail. In recent years, the many advances achieved in the design of pure shift experiments has paved the way to the analysis of a wide range of biological samples with ultra-high resolution. This review leads the reader from the early days of the main pure shift methods that have been successfully developed over the last decades to address complex samples, to the most recent and promising applications of pure shift NMR to the field of NMR-based metabolomics.

A Toolbox for Glutamine Use in Dissolution Dynamic Nuclear Polarization: from Enzymatic Reaction Monitoring to the Study of Cellular Metabolic Pathways and Imaging.

Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D-DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5-13 C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L-asparaginase that has been used as an anti-metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4-13 C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E. coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.

UCP2 silencing restrains leukemia cell proliferation through glutamine metabolic remodeling.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from early T cell progenitors. Since relapsed T-ALL is associated with a poor prognosis improving initial treatment of patients is essential to avoid resistant selection of T-ALL. During initiation, development, metastasis and even in response to chemotherapy, tumor cells face strong metabolic challenges. In this study, we identify mitochondrial UnCoupling Protein 2 (UCP2) as a tricarboxylic acid (TCA) cycle metabolite transporter controlling glutamine metabolism associated with T-ALL cell proliferation. In T-ALL cell lines, we show that UCP2 expression is controlled by glutamine metabolism and is essential for their proliferation. Our data show that T-ALL cell lines differ in their substrate dependency and their energetic metabolism (glycolysis and oxidative). Thus, while UCP2 silencing decreases cell proliferation in all leukemia cells, it also alters mitochondrial respiration of T-ALL cells relying on glutamine-dependent oxidative metabolism by rewiring their cellular metabolism to glycolysis. In this context, the function of UCP2 in the metabolite export of malate enables appropriate TCA cycle to provide building blocks such as lipids for cell growth and mitochondrial respiration. Therefore, interfering with UCP2 function can be considered as an interesting strategy to decrease metabolic efficiency and proliferation rate of leukemia cells.

Ultrahigh-Resolution NMR with Water Signal Suppression for a Deeper Understanding of the Action of Antimetabolic Drugs on Diffuse Large B-Cell Lymphoma.

Ultrahigh-resolution NMR has recently attracted considerable attention in the field of complex samples analysis. Indeed, the implementation of broadband homonuclear decoupling techniques has allowed us to greatly simplify crowded 1H spectra, yielding singlets for almost every proton site from the analyzed molecules. Pure shift methods have notably shown to be particularly suitable for deciphering mixtures of metabolites in biological samples. Here, we have successfully implemented a new pure shift pulse sequence based on the PSYCHE method, which incorporates a block for solvent suppression that is suitable for metabolomics analysis. The resulting experiment allows us to record ultrahigh-resolution 1D NOESY 1H spectra of biofluids with suppression of the water signal, which is a crucial step for highlighting metabolite mixtures in an aqueous phase. We have successfully recorded pure shift spectra on extracellular media of diffuse large B-cell lymphoma (DLBCL) cells. Despite a lower sensitivity, the resolution of pure shift data was found to be better than that of the standard approach, which provides a more detailed vision of the exo-metabolome. The statistical analyses carried out on the resulting metabolic profiles allow us to successfully highlight several metabolic pathways affected by these drugs. Notably, we show that Kidrolase plays a major role in the metabolic pathways of this DLBCL cell line.

Structural analysis of unstable norbixin isomers guided by pure shift nuclear magnetic resonance.

We report the analysis of complex samples obtained during the microwave irradiation/heating of norbixin, which has been identified as a potential therapeutic target for age-related macular degeneration (AMD). In this context, identifying the different isomers that are obtained during its degradation is of primary importance. However, this characterization is challenging because, on the one hand, some of these isomers are unstable, and on the other hand, the 1 H spectra of these isomeric mixtures are poorly resolved. We could successfully apply 1D pure shift experiments to obtain ultrahigh-resolution 1 H nuclear magnetic resonance (NMR) spectra of the norbixin isomer samples and exploit their information content to analyze complementary 2D NMR data and describe accurately their isomeric composition.

Loss of prion protein control of glucose metabolism promotes neurodegeneration in model of prion diseases.

Corruption of cellular prion protein (PrPC) function(s) at the plasma membrane of neurons is at the root of prion diseases, such as Creutzfeldt-Jakob disease and its variant in humans, and Bovine Spongiform Encephalopathies, better known as mad cow disease, in cattle. The roles exerted by PrPC, however, remain poorly elucidated. With the perspective to grasp the molecular pathways of neurodegeneration occurring in prion diseases, and to identify therapeutic targets, achieving a better understanding of PrPC roles is a priority. Based on global approaches that compare the proteome and metabolome of the PrPC expressing 1C11 neuronal stem cell line to those of PrPnull-1C11 cells stably repressed for PrPC expression, we here unravel that PrPC contributes to the regulation of the energetic metabolism by orienting cells towards mitochondrial oxidative degradation of glucose. Through its coupling to cAMP/protein kinase A signaling, PrPC tones down the expression of the pyruvate dehydrogenase kinase 4 (PDK4). Such an event favors the transfer of pyruvate into mitochondria and its conversion into acetyl-CoA by the pyruvate dehydrogenase complex and, thereby, limits fatty acids ß-oxidation and subsequent onset of oxidative stress conditions. The corruption of PrPC metabolic role by pathogenic prions PrPSc causes in the mouse hippocampus an imbalance between glucose oxidative degradation and fatty acids ß-oxidation in a PDK4-dependent manner. The inhibition of PDK4 extends the survival of prion-infected mice, supporting that PrPSc-induced deregulation of PDK4 activity and subsequent metabolic derangements contribute to prion diseases. Our study posits PDK4 as a potential therapeutic target to fight against prion diseases.

The complex metabolism of poststerone in male rats.

Ecdysteroids are not endogenous to mammals, but are normal components of the food intake of many mammalian species consuming phytoecdysteroid-containing plants. The most frequently encountered phytoecdysteroid is 20-hydroxyecdysone (20E). Several pharmaceutical effects have been observed after ecdysteroid injection or ingestion, but it is not clear to what extent metabolites generated in the mammalian body contribute to these effects. The C21-ecdysteroid poststerone (Post) is a metabolite of 20E in rodents. Post analogues are key intermediates in the metabolism of exogenous ecdysteroids possessing a C20/22-diol. The pharmacokinetics, bioavailability and metabolism of Post have been assessed in male rats after ingestion and injection. The bioavailability of Post is significantly greater than that of 20E and the presence of an efficient entero-hepatic cycle allows Post to be effectively metabolised to a wide range of metabolites which are excreted mainly in the faeces, but also to some extent in the urine. Several of the major metabolites in the bile have been identified unambiguously as 3-epi-poststerone, 16α-hydroxypoststerone, 21-hydroxypoststerone and 3-epi-21-hydroxypoststerone. Conjugates are also present. Parallels are drawn to the metabolism of endogenous vertebrate steroid hormones, to which Post bears more similarity than 20E.

Endometriosis phenotypes are associated with specific serum metabolic profiles determined by proton-nuclear magnetic resonance.

RESEARCH QUESTION: What is the correlation between serum metabolic profile and endometriosis phenotype? DESIGN: A pilot study nestled in a prospective cohort study at a university hospital, including 46 patients with painful endometriosis who underwent surgery and 21 controls who did not have macroscopic endometriotic lesions. Endometriosis was strictly classified into two groups of 23 patients each: endometrioma (OMA) and deep infiltrating endometriosis (DIE). Serum samples were collected before surgery for metabolomic profiling based on proton-nuclear magnetic resonance spectroscopy in combination with statistical approaches. Comparative identification of the metabolites in the serum from endometriosis patients and from controls was carried out, including an analysis according to endometriosis phenotype. RESULTS: The serum metabolic profiles of the endometriosis patients revealed significantly lower concentrations of several amino acids compared with the controls, whereas the concentrations of free fatty acids and ketone bodies were significantly higher. The OMA and the DIE phenotypes each had a specific metabolic profile, with higher concentrations of two ketone bodies in the OMA group, and higher concentrations of free fatty acids and lipids in the DIE group. CONCLUSION: Proton-nuclear magnetic resonance-based metabolomics of serum samples were found to have ample potential for identifying metabolic changes associated with endometriosis phenotypes. This information may improve our understanding of the pathogenesis of endometriosis.

Urinary metabolic profiling of asymptomatic acute intermittent porphyria using a rule-mining-based algorithm.

INTRODUCTION: Metabolomic profiling combines Nuclear Magnetic Resonance spectroscopy with supervised statistical analysis that might allow to better understanding the mechanisms of a disease. OBJECTIVES: In this study, the urinary metabolic profiling of individuals with porphyrias was performed to predict different types of disease, and to propose new pathophysiological hypotheses. METHODS: Urine 1H-NMR spectra of 73 patients with asymptomatic acute intermittent porphyria (aAIP) and familial or sporadic porphyria cutanea tarda (f/sPCT) were compared using a supervised rule-mining algorithm. NMR spectrum buckets bins, corresponding to rules, were extracted and a logistic regression was trained. RESULTS: Our rule-mining algorithm generated results were consistent with those obtained using partial least square discriminant analysis (PLS-DA) and the predictive performance of the model was significant. Buckets that were identified by the algorithm corresponded to metabolites involved in glycolysis and energy-conversion pathways, notably acetate, citrate, and pyruvate, which were found in higher concentrations in the urines of aAIP compared with PCT patients. Metabolic profiling did not discriminate sPCT from fPCT patients. CONCLUSION: These results suggest that metabolic reprogramming occurs in aAIP individuals, even in the absence of overt symptoms, and supports the relationship that occur between heme synthesis and mitochondrial energetic metabolism.

Correction to: Urinary metabolic profiling of asymptomatic acute intermittent porphyria using a rule-mining-based algorithm.

The article Urinary metabolic profiling of asymptomatic acute intermittent porphyria using a rule-mining-based algorithm, written by Margaux Luck, Caroline Schmitt, Neila Talbi, Laurent Gouya, Cédric Caradeuc, Hervé Puy, Gildas Bertho and Nicolas Pallet was originally published Online First without open access. After publication in volume [14], issue [1], Citation ID[10] the author decided to opt for Open Choice and to make the article an open access publication. Therefore, the copyright of the article has been changed to © The Author(s) 2018 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The original article has been corrected.

Real-Time and Non-invasive Monitoring of the Activation of the IRE1α-XBP1 Pathway in Individuals with Hemodynamic Impairment.

Many stressors that are encountered upon kidney injury are likely to trigger endoplasmic reticulum (ER) stress, subsequently activating transcriptional, translational and metabolic reprogramming. Monitoring early cellular adaptive responses engaged after hemodynamic impairment yields may represent a clinically relevant approach. However, a non-invasive method for detecting the ER stress response has not been developed. We combined a metabolomic approach with genetic marker analyses using urine from individuals undergoing scheduled cardiac surgery under cardiopulmonary bypass to investigate the feasibility and significance of monitoring the ER stress response in the kidney. We developed an original method based on fragment analysis that measures urinary levels of the spliced X-box binding protein 1 (sXBP1) mRNA as a proxy of inositol-requiring enzyme 1α (IRE1α) activity because sXBP1 is absolutely sensitive and specific for ER stress. The early engagement of the ER stress response after ischemic stress is critical for protecting against tissue damage, and individuals who mount a robust adaptive response are protected against AKI. The clinical consequences of our findings are of considerable importance because ER stress is involved in numerous conditions that lead to AKI and chronic kidney disease; in addition, the detection of ER stress is straightforward and immediately available in routine practice.

Specific Physical Exercise Improves Energetic Metabolism in the Skeletal Muscle of Amyotrophic-Lateral- Sclerosis Mice.

Amyotrophic Lateral Sclerosis is an adult-onset neurodegenerative disease characterized by the specific loss of motor neurons, leading to muscle paralysis and death. Although the cellular mechanisms underlying amyotrophic lateral sclerosis (ALS)-induced toxicity for motor neurons remain poorly understood, growing evidence suggest a defective energetic metabolism in skeletal muscles participating in ALS-induced motor neuron death ultimately destabilizing neuromuscular junctions. In the present study, we report that a specific exercise paradigm, based on a high intensity and amplitude swimming exercise, significantly improves glucose metabolism in ALS mice. Using physiological tests and a biophysics approach based on nuclear magnetic resonance (NMR), we unexpectedly found that SOD1(G93A) ALS mice suffered from severe glucose intolerance, which was counteracted by high intensity swimming but not moderate intensity running exercise. Furthermore, swimming exercise restored the highly ALS-sensitive tibialis muscle through an autophagy-linked mechanism involving the expression of key glucose transporters and metabolic enzymes, including GLUT4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Importantly, GLUT4 and GAPDH expression defects were also found in muscles from ALS patients. Moreover, we report that swimming exercise induced a triglyceride accumulation in ALS tibialis, likely resulting from an increase in the expression levels of lipid transporters and biosynthesis enzymes, notably DGAT1 and related proteins. All these data provide the first molecular basis for the differential effects of specific exercise type and intensity in ALS, calling for the use of physical exercise as an appropriate intervention to alleviate symptoms in this debilitating disease.