Operation of a TCA cycle subnetwork in the mammalian nucleus.

Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.

Prior Signal Acquisition Software Versions for Orbitrap Underestimate Low Isobaric Mass Tag Intensities, Without Detriment to Differential Abundance Experiments.

Tandem mass tags (TMTs) enable simple and accurate quantitative proteomics for multiplexed samples by relative quantification of tag reporter ions. Orbitrap quantification of reporter ions has been associated with a characteristic notch region in intensity distribution, within which few reporter intensities are recorded. This has been resolved in version 3 of the instrument acquisition software Tune. However, 47% of Orbitrap Fusion, Lumos, or Eclipse submissions to PRIDE were generated using prior software versions. To quantify the impact of the notch on existing quantitative proteomics data, we generated a mixed species benchmark and acquired quantitative data using Tune versions 2 and 3. Intensities below the notch are predominantly underestimated with Tune version 2, leading to overestimation of the true differences in intensities between samples. However, when summarizing reporter ion intensities to higher-level features, such as peptides and proteins, few features are significantly affected. Targeted removal of spectra with reporter ion intensities below the notch is not beneficial for differential peptide or protein testing. Overall, we find that the systematic quantification bias associated with the notch is not detrimental for a typical proteomics experiment.

Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.

A Surprisingly Simple Electrostatic Model Explains Bent Versus Linear Structures in M(+)-RG2 Species (M = Group 1 Metal, Li-Fr; RG = Rare Gas, He-Rn).

It is found that a simple electrostatic model involving competition between the attractive dispersive interaction and induced-dipole repulsion between the two RG atoms performs extremely well in rationalizing the M(+)-RG2 geometries, where M = group 1 metal and RG = rare gas. The Li(+)-RG2 and Na(+)-RG2 complexes have previously been found to exhibit quasilinear or linear minimum-energy geometries, with the Na(+)-RG2 complexes having an additional bent local minimum [A. Andrejeva, A. M. Gardner, J. B. Graneek, R. J. Plowright, W. H. Breckenridge, T. G. Wright, J. Phys. Chem. A, 2013, 117, 13578]. In the present work, the geometries for M = K-Fr are found to be bent. A simple electrostatic model explains these conclusions and is able to account almost quantitatively for the binding energy of the second RG atom, as well as the form of the angular potential, for all 36 titular species. Additionally, results of population analyses are presented together with orbital contour plots; combined with the success of the electrostatic model, the expectation that these complexes are all physically bound is confirmed.

Assignment of the vibrations of the S0, S1, and D0 (+) states of perhydrogenated and perdeuterated isotopologues of chlorobenzene.

We report vibrationally resolved spectra of the S1←S0 transition of chlorobenzene using resonance-enhanced multiphoton ionization spectroscopy. We study chlorobenzene-h5 as well as its perdeuterated isotopologue, chlorobenzene-d5. Changes in the form of the vibrational modes between the isotopologues and also between the S0 and S1 electronic states are discussed for each species. Vibrational bands are assigned utilizing quantum chemical calculations, previous experimental results, and isotopic shifts, including those between the (35)Cl and (37)Cl isotopologues. Previous work and assignments of the S1 spectra are discussed. Additionally, the vibrations in the ground state cation, D0 (+), are considered, since these have also been used by previous workers in assigning the excited neutral state spectra.

Theoretical Study of M(+)-RG2: (M(+) = Ca, Sr, Ba, and Ra; RG = He-Rn).

Ab initio calculations were employed to investigate M(+)-RG2 species, where M(+) = Ca, Sr, Ba, and Ra and RG = He-Rn. Geometries have been optimized, and cuts through the potential energy surfaces containing each global minimum have been calculated at the MP2 level of theory, employing triple-ζ quality basis sets. The interaction energies for these complexes were calculated employing the RCCSD(T) level of theory with quadruple-ζ quality basis sets. Trends in binding energies, De, equilibrium bond lengths, Re, and bond angles are discussed and rationalized by analyzing the electronic density. Mulliken, natural population, and atoms-in-molecules (AIM) population analyses are presented. It is found that some of these complexes involving the heavier group 2 metals are bent whereas others are linear, deviating from observations for the corresponding Be and Mg metal-containing complexes, which have all previously been found to be bent. The results are discussed in terms of orbital hybridization and the different types of interaction present in these species.

Resonance-enhanced multiphoton ionization (REMPI) spectroscopy of bromobenzene and its perdeuterated isotopologue: Assignment of the vibrations of the S(0), S(1), and D(0)(+) states of bromobenzene and the S(0) and D(0)(+) states of iodobenzene.

We report vibrationally resolved spectra of the S1←S0 transition of bromobenzene using resonance-enhanced multiphoton ionization spectroscopy. We study bromobenzene-h5 as well as its perdeuterated isotopologue, bromobenzene-d5. The form of the vibrational modes between the isotopologues and also between the S0 and S1 electronic states is discussed for each species, allowing assignment of the bands to be achieved and the activity between states and isotopologues to be established. Vibrational bands are assigned utilizing quantum chemical calculations, previous experimental results, and isotopic shifts. Previous work and assignments of the S1 spectra are discussed. Additionally, the vibrations in the ground state cation, D0 (+), are considered, since these have also been used by previous workers in assigning the excited neutral state spectra. We also examine the vibrations of iodobenzene in the S0 and D0 (+) states and comment on the previous assignments of these. In summary, we have been able to assign the corresponding vibrations across the whole monohalobenzene series of molecules, in the S0, S1, and D0 (+) states, gaining insight into vibrational activity and vibrational couplings.

Vibrations of the S1 state of fluorobenzene-h5 and fluorobenzene-d5 via resonance-enhanced multiphoton ionization (REMPI) spectroscopy.

We report resonance-enhanced multiphoton ionization spectra of the isotopologues fluorobenzene-h5 and fluorobenzene-d5. By making use of quantum chemical calculations, the changes in the wavenumber of the vibrational modes upon deuteration are examined. Additionally, the mixing of vibrational modes both between isotopologues and also between the two electronic states is discussed. The isotopic shifts lead to dramatic changes in the appearance of the spectrum as vibrations shift in and out of Fermi resonance. Assignments of the majority of the fluorobenzene-d5 observed bands are provided, aided by previous results on fluorobenzene-h5.

Theoretical study of M(+)-RG2 (M+ = Li, Na, Be, Mg; RG = He-Rn).

Ab initio calculations were employed to determine the geometry (MP2 level), and dissociation energies [MP2 and RCCSD(T) levels], of the M(IIa)(+)-RG2 species, where M(IIa) is a group 2 metal, Be or Mg, and RG is a rare gas (He-Rn). We compare the results with similar calculations on M(Ia)(+)-RG2, where M(Ia) is a group 1 metal, Li or Na. It is found that the complexes involving the group 1 metals are linear (or quasilinear), whereas those involving the group 2 metals are bent. We discuss these results in terms of hybridization and the various interactions in these species. Trends in binding energies, D(e), bond lengths, and bond angles are discussed. We compare the energy required for the removal of a single RG atom from M(+)-RG2 (D(e2)) with that of the dissociation energy of M(+)-RG (D(e1)); some complexes have D(e2) > D(e1), some have D(e2) < D(e1), and some have values that are about the same. We also present relaxed angular cuts through a selection of potential energy surfaces. The trends observed in the geometries and binding energies of these complexes are discussed. Mulliken, natural population, and atoms-in-molecules (AIM) population analyses are performed, and it is concluded that the AIM method is the most reliable, giving results that are in line with molecular orbital diagrams and contour plots; unphysical amounts of charge transfer are suggested by the Mulliken and natural population approaches.

Interaction of the NO 3pπ Rydberg state with Ar: potential energy surfaces and spectroscopy.

We present the experimental and simulated (2+1) REMPI spectrum of the C(2)Π state of the NO-Ar complex, in the vicinity of the 3p Rydberg state of NO. Two Rydberg states of NO are expected in this energy region: the C(2)Π (3pπ) and D(2)Σ(+) (3pσ) states, and we concentrate on the former here. When the C(2)Π (3pπ) state interacts with Ar at nonlinear orientations, the symmetry is lowered to C(s), splitting the degeneracy of the (2)Π state to yield C((2)A") and C((2)A') states. For these two states of NO-Ar, we calculate potential energy surfaces using second order Møller-Plesset perturbation theory, exploiting a procedure to converge the reference Hartree-Fock wavefunction to describe the excited states, the maximum overlap method. The bound rovibrational states obtained from the surfaces are used to simulate the electronic spectrum, which is in excellent agreement with experiment, providing assignments for the observed spectral lines from the calculated rovibrational wavefunctions.