Equity, Diversity, and Inclusion Climate Assessment Activities: Development and Strategic Use in Diversity Action Plans

This project report describes climate assessment activities in support of the development of a college-level diversity, equity, and inclusion (EDI) action plan. Elements of the climate assessment activities are described along with their purpose and rationale for inclusion. Recommendations are made for libraries to design and deploy their own EDI assessment activities with the goal of developing robust action plans supportive of inclusive excellence

Local protein kinase A action proceeds through intact holoenzymes

Hormones can transmit signals through adenosine 3’,5’-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory (RII) subunits with A-kinase anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C sub-assemblies within 150 to 250Å of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP. Chemical-biology and live-cell imaging techniques revealed that catalytically active PKA holoenzymes remained intact within the cytoplasm. In contrast, little, if any PKA activity was detected in the nucleus. Hence the parameters of anchored PKA holoenzyme action are much more restricted than originally anticipated

Covalent Aurora A regulation by the metabolic integrator coenzyme A

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues

Roadmap on dynamics of molecules and clusters in the gas phase

This roadmap article highlights recent advances, challenges and future prospects in studies of the dynamics of molecules and clusters in the gas phase. It comprises nineteen contributions by scientists with leading expertise in complementary experimental and theoretical techniques to probe the dynamics on timescales spanning twenty order of magnitudes, from attoseconds to minutes and beyond, and for systems ranging in complexity from the smallest (diatomic) molecules to clusters and nanoparticles. Combining some of these techniques opens up new avenues to unravel hitherto unexplored reaction pathways and mechanisms, and to establish their significance in, e.g. radiotherapy and radiation damage on the nanoscale, astrophysics, astrochemistry and atmospheric science

Mass spectrometry-based approaches to study lanthanides and lanthanide-dependent proteins in the phyllosphere

Rare-earth elements (REEs) were recently discovered to be biologically significant. The finding was originally made with the methanol dehydrogenase XoxF, which depends on REEs for its activity, and reports of lanthanide-utilizing bacteria have since expanded. Environmental proteomics allows the identification of proteins specifically induced by the presence of lanthanides or can provide insights into the preferred use of lanthanide-dependent and -independent isoenzymes, for example. Here we describe protocols for the growth and subsequent mass spectrometry-based proteome analysis of bacteria obtained from controlled artificial media and from the phyllosphere of the model plant Arabidopsis thaliana. In addition, the use of inductively coupled plasma mass spectrometry (ICP-MS) is described for the quantification of REEs in biological samples.ISSN:15577988ISSN:0076-687

Use of rare-earth elements in the phyllosphere colonizer Methylobacterium extorquens PA1

Until recently, rare‐earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase XoxF that strictly relies on REEs for its activity. Some methylotrophs only contain xoxF, while others, including the model phyllosphere colonizer Methylobacterium extorquens PA1, harbor this gene in addition to mxaFI encoding a Ca2+‐dependent enzyme. Here we found that REEs induce the expression of xoxF in M. extorquens PA1, while repressing mxaFI, suggesting that XoxF is the preferred methanol dehydrogenase in the presence of sufficient amounts of REE. Using reporter assays and a suppressor screen, we found that lanthanum (La3+) is sensed both in a XoxF‐dependent and independent manner. Furthermore, we investigated the role of REEs during Arabidopsis thaliana colonization. Element analysis of the phyllosphere revealed the presence of several REEs at concentrations up to 10 μg per g dry weight. Complementary proteome analyses of M. extorquens PA1 identified XoxF as a top induced protein in planta and a core set of La3+‐regulated proteins under defined artificial media conditions. Among these was a REE‐binding protein that is encoded next to a gene for a TonB‐dependent transporter. The latter was essential for REE‐dependent growth on methanol indicating chelator‐assisted uptake of REEs

Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa-protein without functional annotation. The protein comprises two characteristic PepSY domains and we thus termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications towards the sustainable purification and separation of rare-earth elements.ISSN:0021-9258ISSN:1083-351

Conformer-selective photoelectron spectroscopy of alpha-lactalbumin derived multianions in the gas phase

International audienceWe have recorded conformer-selective, gas-phase photoelectron spectra of a-lactalbumin derived multianions generated by electrospraying solutions of both the native protein and its denatured form (as prepared by breaking the sulfur-sulfur bonds by chemical reduction). Three different groups of gas-phase multianion conformers have been observed and characterized. Highly-folded and partially-unfolded structures are obtained from solutions of the native protein. Only highly-elongated conformers are observed upon electrospraying the denatured protein. Adiabatic detachment energies were determined at several negative charge states for each conformer group. In comparison to highly-elongated conformations, highly-folded structures show a steeper decrease of electron binding energy with increasing negative charge. By comparing experimental detachment energies for highly-elongated structures with the predictions of a simple electrostatic model calculation, we have determined the effective dielectric shielding constant

Isomer-Selected Photoelectron Spectroscopy of Isolated DNA Oligonucleotides: Phosphate and Nucleobase Deprotonation at High Negative Charge States

Fractionation according to ion mobility and mass-to-charge ratio has been used to select individual isomers of deprotonated DNA oligonucleotide multianions for subsequent isomer-resolved photoelectron spectroscopy (PES) in the gas phase. Isomer-resolved PE spectra have been recorded for tetranucleotides, pentanucleotides, and hexanucleotides. These were studied primarily in their highest accessible negative charge states (3–, 4–, and 5–, respectively), as provided by electrospraying from room temperature solutions. In particular, the PE spectra obtained for pentanucleotide tetraanions show evidence for two coexisting classes of gas-phase isomeric structures. We suggest that these two classes comprise: (i) species with excess electrons localized exclusively at deprotonated phosphate backbone sites and (ii) species with at least one deprotonated base (in addition to several deprotonated phosphates). By permuting the sequence of bases in various [A_5–<i>x</i>T_<i>x</i>]^4– and [GT₄]^4– pentanucleotides, we have established that the second type of isomer is most likely to occur if the deprotonated base is located at the first or last position in the sequence. We have used a combination of molecular mechanics and semiempirical calculations together with a simple electrostatic model to explore the photodetachment mechanism underlying our photoelectron spectra. Comparison of predicted to measured photoelectron spectra suggests that a significant fraction of the detected electrons originates from the DNA bases (both deprotonated and neutral)