Thanatometabolomics: introducing NMR-based metabolomics to identify metabolic biomarkers of the time of death.

Death is the permanent cessation of the critical functions of the organism as a whole. However, the shutdown of a complex biological organism does not abruptly terminate at time of death. New high-throughput technologies allow the systematic investigation of the biochemical modulations occurring after death. Recent genomics studies have demonstrated that genes remain active after death, triggering upregulation of some genes and initiating feedback loops. These genes were mostly involved in pathways related to immunity, inflammation and cancer. These genetic modulations suggest many biochemical events persist after death, which can be captured using a metabolomics approach. This proof of concept work aimed to determine whether NMR spectroscopy could identify metabolomics changes occurring after death, and characterise the nature of these metabolomics modulations. High-resolution H-NMR spectroscopy was applied to six biological matrices: heart, kidney, liver, spleen, skin and white adipose tissue of ten adult mice at three different type points. Forty-three metabolites were associated with post mortem metabolomics modulations. Kidney, heart and spleen showed the highest metabolic perturbations. Conversely, skin and white adipose tissue were the least altered matrices. Early metabolic modulations were associated with energy metabolism and DNA synthesis, by contrast, late metabolomics modulations were associated with microbial metabolism. NMR has proven potential to determine the time of death based on post-mortem metabolomics modulations. This could be useful in the context of transplants, forensic studies and as internal quality control in metabolomics studies. Further investigations are required to validate these findings in humans in order to determine which compounds robustly reflect post-mortem metabolic fluctuations to accurately determine the time of death

NMR metabolomics identifies over 60 biomarkers associated with Type II Diabetes impairment in db/db mice

The rapid expansion of Type 2 Diabetes (T2D), that currently affects 90% of people suffering from diabetes, urges us to develop a better understanding of the metabolic processes involved in the disease process in order to develop better therapies. The most commonly used model for T2D research is the db/db (BKS.Cg-Dock7  +/+ Lepr /J) mouse model. Yet, a systematic H NMR based metabolomics characterisation of most tissues in this animal model has not been published. Here, we provide a systematic organ-specific metabolomics analysis of this widely employed model using NMR spectroscopy. The aim of this study was to characterise the metabolic modulations associated with T2D in db/db mice in 18 relevant biological matrices. High-resolution H-NMR and 2D-NMR spectroscopy were applied to 18 biological matrices of 12 db/db mice (WT control n = 6, db/db = 6) aged 22 weeks, when diabetes is fully established. 61 metabolites associated with T2D were identified. Kidney, spleen, eye and plasma were the biological matrices carrying the largest metabolomics modulations observed in established T2D, based on the total number of metabolites that showed a statistical difference between the diabetic and control group in each tissue (16 in each case) and the strength of the O-PLS DA model for each tissue. Glucose and glutamate were the most commonly associated metabolites found significantly increased in nine biological matrices. Investigated sections where no increase of glucose was associated with T2D include all intestinal segments (i.e. duodenum, jejunum, ileum and colon). Microbial co-metabolites such as acetate and butyrate, used as carbon sources by the host, were identified in excess in the colonic tissues of diabetic individuals. The metabolic biomarkers identified using H NMR-based metabolomics will represent a useful resource to explore metabolic pathways involved in T2D in the db/db mouse model

Conformational Dynamics of Asparagine at Coiled-Coil Interfaces

Coiled coils (CCs) are among the best-understood protein folds. Nonetheless, there are gaps in our knowledge of CCs. Notably, CCs are likely to be structurally more dynamic than often considered. Here, we explore this in an abundant class of CCs, parallel dimers, focusing on polar asparagine (Asn) residues in the hydrophobic interface. It is well documented that such inclusions discriminate between different CC oligomers, which has been rationalized in terms of whether the Asn can make side-chain hydrogen bonds. Analysis of parallel CC dimers in the Protein Data Bank reveals a variety of Asn side-chain conformations, but not all of these make the expected inter-side-chain hydrogen bond. We probe the structure and dynamics of a <i>de novo</i>-designed coiled-coil homodimer, CC-Di, by multidimensional nuclear magnetic resonance spectroscopy, including model-free dynamical analysis and relaxation–dispersion experiments. We find dynamic exchange on the millisecond time scale between Asn conformers with the side chains pointing into and out of the core. We perform molecular dynamics simulations that are consistent with this, revealing that the side chains are highly dynamic, exchanging between hydrogen-bonded-paired conformations in picoseconds to nanoseconds. Combined, our data present a more dynamic view for Asn at CC interfaces. Although inter-side-chain hydrogen bonding states are the most abundant, Asn is not always buried or engaged in such interactions. Because interfacial Asn residues are key design features for modulating CC stability and recognition, these further insights into how they are accommodated within CC structures will aid their predictive modeling, engineering, and design

Résonance magnétique nucléaire du Xénon 129 polarisé pour des applications biomédicales ou dans des systèmes à faible densité de spins

La recherche biomédicale par résonance magnétique nucléaire (RMN) s'est vue ouvrir de nouvelles perspectives avec l'utilisation des gaz nobles hyperpolarisés (Hélium et Xénon). Leur polarisation nucléaire peut être augmentée par la méthode de pompage optique, qui été mise en œuvre pendant le travail de thèse. Le gain en polarisation, pouvant aller jusqu'à cinq ordres de grandeurs, est directement répercuté sur le signal de RMN. Un tel gain de signal promet une augmentation de sensibilité qui élargit considérablement le champ des applications de l'imagerie et de la spectroscopie par RMN.Des études de faisabilité pour des applications du Xénon hyperpolarisé sont en cours pour deux domaines d'expériences. Le premier porte sur l'étude de certaines maladies neurodégénératives, comme les maladies démyélinisantes. En effet, le Xénon est un gaz qui se concentre dans les tissus riches en lipides tels que ceux du système nerveux central. Mais pour conserver assez de signal, il faut que le transport jusqu'à l'organe cible préserve autant que possible l'hyperpolarisation (distance courte et vectorisation du Xénon). Une séquence de RMN à fort rapport signal à bruit a été mise en œuvre pour l'étude du Xénon non hyperpolarisé dans les vecteurs biocompatibles pressentis : la séquence Steady State Free Precession. Cette séquence permet d'obtenir les paramètres importants de RMN avant l'utilisation du Xénon hyperpolarisé. On simplifie ainsi les protocoles d'expériences. La deuxième voie de recherche prévue est l'imagerie du Xénon hyperpolarisé, pour essayer de visualiser les déformations des mousses aqueuses en trois dimensions. En effet, la structure de celles-ci permet d'élaborer des modèles expérimentaux des matériaux granulaires.Les résultats obtenus faciliteront l'utilisation du Xénon hyperpolarisé comme traceur des milieux biologiques et des systèmes physiques peu denses.Hyperpolarized noble gases (helium and xenon) have shown new directions in nuclear magnetic resonance (NMR) biomedical research. The nuclear polarization can be increased by spin-exchange optical pumping, whose implementation was part of the thesis work. The polarization gain, up to five orders, is directly seen on the NMR signal. This lead to an increase of sensitivity therefore broadening the field of NMR imaging and spectroscopy applications.Two hyperpolarized xenon applications feasability studies are currently running. The first concerns the study of neurodegenerative deseases, like demyelinisating pathology. One of the properties of the gaseous Xenon is to concentrate on lipidic tissues, like those of the central nervous system. An efficient method to deliver the gas while maintaining the high level of polarization is to use carrier agents. The steady state free precession sequence have been implemented for non-hyperpolarized xenon studies on bio-compatible carriers. The main NMR parameters can then be known prior to hyperpolarized xenon experiments. Thus, time and hyperpolarized xenon are saved. The second feasability study is on hyperpolarized xenon three dimensions imaging on aqueous bubbles under constraints, because their structures allow experiemental modeling of granular materials. Results will make easier the use of hyperpolarized xenon as biological and diluted physical systems tracer.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

The Structural Properties in Solution of the Intrinsically Mixed Folded Protein Ataxin-3

It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions that can both contribute to function. Although equally interesting, the disordered regions are difficult to study, because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. NMR spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, a task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe the structural properties of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. Ataxin-3 is a 42-kDa protein containing a globular N-terminal Josephin domain and a C-terminal tail that comprises 13 polyglutamine repeats within a low complexity region. We developed a strategy that allowed us to achieve 87% assignment of the NMR spectrum using a mixed protocol based on high-dimensionality, high-resolution experiments and different labeling schemes. Thanks to the almost complete spectral assignment, we proved that the C-terminal tail is flexible, with extended helical regions, and interacts only marginally with the rest of the protein. We could also, for the first time to our knowledge, observe the structural propensity of the polyglutamine repeats within the context of the full-length protein and show that its structure is stabilized by the preceding region

Controlling the dynamics of the Nek2 leucine zipper by engineering of "kinetic" disulphide bonds

<div><p>Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2’s catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG^‡) and maximal rate constant (k’_ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of ‘kinetic’ disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.</p></div

Mechanism of β-actin mRNA Recognition by ZBP1

Zipcode binding protein 1 (ZBP1) is an oncofetal RNA-binding protein that mediates the transport and local translation of β-actin mRNA by the KH3-KH4 di-domain, which is essential for neuronal development. The high-resolution structures of KH3-KH4 with their respective target sequences show that KH4 recognizes a non-canonical GGA sequence via an enlarged and dynamic hydrophobic groove, whereas KH3 binding to a core CA sequence occurs with low specificity. A data-informed kinetic simulation of the two-step binding reaction reveals that the overall reaction is driven by the second binding event and that the moderate affinities of the individual interactions favor RNA looping. Furthermore, the concentration of ZBP1, but not of the target RNA, modulates the interaction, which explains the functional significance of enhanced ZBP1 expression during embryonic development

Cluster and Fold Stability of <i>E. coli</i> ISC-Type Ferredoxin

<div><p>Iron-sulfur clusters are essential protein prosthetic groups that provide their redox potential to several different metabolic pathways. Formation of iron-sulfur clusters is assisted by a specialised machine that comprises, among other proteins, a ferredoxin. As a first step to elucidate the precise role of this protein in cluster assembly, we have studied the factors governing the stability and the dynamic properties of <i>E. coli</i> ferredoxin using different spectroscopic techniques. The cluster-loaded protein is monomeric and well structured with a flexible C-terminus but is highly oxygen sensitive so that it readily loses the cluster leading to an irreversible unfolding under aerobic conditions. This process is slowed down by reducing conditions and high ionic strengths. NMR relaxation experiments on the cluster-loaded protein also show that, once the cluster is in place, the protein forms a globular and relatively rigid domain. These data indicate that the presence of the iron-sulfur cluster is the switch between a functional and a non-functional state.</p></div

Structural and dynamic characterization of Fdx.

<p>Chemical Shift Index analysis of CA, CB and C’ of holo-Fdx. Assigned residues are coloured green. Red “lollipops” denote helical chemical shifts, blue “lollipops” denote β sheet chemical shifts. Secondary structure is shown under the CSI plot where there is a consensus. B) NMR relaxation parameters.</p

¹⁵N HSQC of holo-Fdx showing the assigned residues.

<p>SC denotes the tryptophan indole side chain N^ε1H^ε1 group. Folded peaks are shown in red.</p