Understanding nucleic acid-ion interactions.

Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.

Local extracellular K+ in cortex regulates norepinephrine levels, network state, and behavioral output.

Extracellular potassium concentration ([K+]e) is known to increase as a function of arousal. [K+]e is also a potent modulator of transmitter release. Yet, it is not known whether [K+]e is involved in the neuromodulator release associated with behavioral transitions. We here show that manipulating [K+]e controls the local release of monoaminergic neuromodulators, including norepinephrine (NE), serotonin, and dopamine. Imposing a [K+]e increase is adequate to boost local NE levels, and conversely, lowering [K+]e can attenuate local NE. Electroencephalography analysis and behavioral assays revealed that manipulation of cortical [K+]e was sufficient to alter the sleep-wake cycle and behavior of mice. These observations point to the concept that NE levels in the cortex are not solely determined by subcortical release, but that local [K+]e dynamics have a strong impact on cortical NE. Thus, cortical [K+]e is an underappreciated regulator of behavioral transitions.

Specifically adsorbed ferrous ions modulate interfacial affinity for high-rate ammonia electrosynthesis from nitrate in neutral media.

The electrolysis of nitrate reduction to ammonia (NRA) is promising for obtaining value-added chemicals and mitigating environmental concerns. Recently, catalysts with high-performance ammonia synthesis from nitrate has been achieved under alkaline or acidic conditions. However, NRA in neutral solution still suffers from the low yield rate and selectivity of ammonia due to the low binding affinity and nucleophilicity of NO3-. Here, we confirmed that the in-situ-generated Fe(II) ions existed as specifically adsorbed cations in the inner Helmholtz plane (IHP) with a low redox potential. Inspired by this, a strategy (Fe-IHP strategy) was proposed to enhance NRA activity by tuning the affinity of the electrode-electrolyte interface. The specifically adsorbed Fe(II) ions [SA-Fe(II)] greatly alleviated the electrostatic repulsion around the interfaceresulting in a 10-fold lower in the adsorption-free energy of NO3- when compared to the case without SA-Fe(II). Meanwhile, the modulated interface accelerated the kinetic mass transfer process by 25 folds compared to the control. Under neutral conditions, a Faraday efficiency of 99.6%, a selectivity of 99%, and an extremely high NH3 yield rate of 485.8 mmol h-1 g-1 FeOOH were achieved. Theoretical calculations and in-situ Raman spectroscopy confirmed the electron-rich state of the SA-Fe(II) donated to p orbitals of N atom and favored the hydrogenation of *NO to *NOH for promoting the formation of high-selectivity ammonia. In sum, these findings complement the textbook on the specific adsorption of cations and provide insights into the design of low-cost NRA catalysts with efficient ammonia synthesis.

The discovery of a catalytic RNA within RNase P and its legacy.

Sidney Altman's discovery of the processing of one RNA by another RNA that acts like an enzyme was revolutionary in biology and the basis for his sharing the 1989 Nobel Prize in Chemistry with Thomas Cech. These breakthrough findings support the key role of RNA in molecular evolution, where replicating RNAs (and similar chemical derivatives) either with or without peptides functioned in protocells during the early stages of life on Earth, an era referred to as the RNA world. Here, we cover the historical background highlighting the work of Altman and his colleagues and the subsequent efforts of other researchers to understand the biological function of RNase P and its catalytic RNA subunit and to employ it as a tool to downregulate gene expression. We primarily discuss bacterial RNase P-related studies but acknowledge that many groups have significantly contributed to our understanding of archaeal and eukaryotic RNase P, as reviewed in this special issue and elsewhere.

Crystal structure and mechanistic studies of the PPM1D serine/threonine phosphatase catalytic domain.

Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short ß-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics.

The Ca2+-MdCRF4-MdWRKY9 module negatively affects apple fruit watercore formation by suppressing the transcription of MdSOT2.

Watercore is a common physiological disease of Rosaceae plants, such as apples (Malus domestica), usually occurring during fruit ripening. Apple fruit with watercore symptoms is prone to browning and rotting, thus losing commercial viability. Sorbitol and calcium ions are considered key factors affecting watercore occurrence in apples. However, the mechanism by which they affect the occurrence of watercore remains unclear. Here, we identified that the transcription factor MdWRKY9 directly binds to the promoter of MdSOT2, positively regulates the transcription of MdSOT2, increases sorbitol content in fruit, and promotes watercore occurrence. Additionally, MdCRF4 can directly bind to MdWRKY9 and MdSOT2 promoters, positively regulating their expression. Since calcium ions can induce the ubiquitination and degradation of the transcription factor MdCRF4, they can inhibit the transcription of MdWRKY9 and MdSOT2 by degrading MdCRF4, thereby reducing the sorbitol content in fruit and inhibiting the occurrence of fruit watercore disease. Our data sheds light on how calcium ions mitigate watercore in fruit, providing molecular-level insights to enhance fruit quality artificially.

Structure and ion-dependent folding of k-junctions.

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of Arabidopsis and Escherichia coli thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the Arabidopsis and E. coli riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the E. coli TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.

SIRT3 Regulates Clearance of Apoptotic Cardiomyocytes by Deacetylating Frataxin.

BACKGROUND: Efferocytosis is an activity of macrophages that is pivotal for the resolution of inflammation in hypertension. The precise mechanism by which macrophages coordinate efferocytosis and internalize apoptotic cardiomyocytes remains unknown. The aim of this study was to determine whether SIRT3 (sirtuin-3) is required for both apoptotic cardiomyocyte engulfment and anti-inflammatory responses during efferocytosis. METHODS: We generated myeloid SIRT3 knockout mice and FXN (frataxin) knock-in mice carrying an acetylation-defective lysine to arginine K189R mutation (FXNK189R). The mice were given Ang II (angiotensin II) infusion for 7 days. We analyzed cardiac macrophages' mitochondrial iron levels, efferocytosis activity, and phenotype both in vivo and in vitro. RESULTS: We showed that SIRT3 deficiency exacerbated Ang II-induced downregulation of the efferocytosis receptor MerTK (c-Mer tyrosine kinase) and proinflammatory cytokine production, accompanied by disrupted mitochondrial iron homeostasis in cardiac macrophages. Quantitative acetylome analysis revealed that SIRT3 deacetylated FXN at lysine 189. Ang II attenuated SIRT3 activity and enhanced the acetylation level of FXNK189. Acetylated FXN further reduced the synthesis of ISCs (iron-sulfur clusters), resulting in mitochondrial iron accumulation. Phagocytic internalization of apoptotic cardiomyocytes increased myoglobin content, and derived iron ions promoted mitochondrial iron overload and lipid peroxidation. An iron chelator deferoxamine improved the levels of MerTK and efferocytosis, thereby attenuating proinflammatory macrophage activation. FXNK189R mice showed improved macrophage efferocytosis, reduced cardiac inflammation, and suppressed cardiac fibrosis. CONCLUSIONS: The SIRT3-FXN axis has the potential to resolve cardiac inflammation by increasing macrophage efferocytosis and anti-inflammatory activities.

Structure, function, and evolution of the metal-binding domain in the nucleosome.

The eukaryotic nucleosome, the basic unit of chromatin, is thermodynamically stable and plays critical roles in the cell, including the maintenance of DNA topology and regulation of gene expression. At its C2 axis of symmetry, the nucleosome exhibits a domain that can coordinate divalent metal ions. This article discusses the roles of the metal-binding domain in the nucleosome structure, function, and evolution.

Oxonium Ion-Guided Optimization of Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro.

Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon-stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

Dissecting Detergent-Insoluble Proteome in Alzheimer's Disease by TMTc-Corrected Quantitative Mass Spectrometry.

Protein aggregation of amyloid-ß peptides and tau are pathological hallmarks of Alzheimer's disease (AD), which are often resistant to detergent extraction and thus enriched in the insoluble proteome. However, additional proteins that coaccumulate in the detergent-insoluble AD brain proteome remain understudied. Here, we comprehensively characterized key proteins and pathways in the detergent-insoluble proteome from human AD brain samples using differential extraction, tandem mass tag (TMT) labeling, and two-dimensional LC-tandem mass spectrometry. To improve quantification accuracy of the TMT method, we developed a complement TMT-based strategy to correct for ratio compression. Through the meta-analysis of two independent detergent-insoluble AD proteome datasets (8914 and 8917 proteins), we identified 190 differentially expressed proteins in AD compared with control brains, highlighting the pathways of amyloid cascade, RNA splicing, endocytosis/exocytosis, protein degradation, and synaptic activity. To differentiate the truly detergent-insoluble proteins from copurified background during protein extraction, we analyzed the fold of enrichment for each protein by comparing the detergent-insoluble proteome with the whole proteome from the same AD samples. Among the 190 differentially expressed proteins, 84 (51%) proteins of the upregulated proteins (n = 165) were enriched in the insoluble proteome, whereas all downregulated proteins (n = 25) were not enriched, indicating that they were copurified components. The vast majority of these enriched 84 proteins harbor low-complexity regions in their sequences, including amyloid-ß, Tau, TARDBP/TAR DNA-binding protein 43, SNRNP70/U1-70K, MDK, PTN, NTN1, NTN3, and SMOC1. Moreover, many of the enriched proteins in AD were validated in the detergent-insoluble proteome by five steps of differential extraction, proteomic analysis, or immunoblotting. Our study reveals a resource list of proteins and pathways that are exclusively present in the detergent-insoluble proteome, providing novel molecular insights to the formation of protein pathology in AD.

Defining the challenges of Li extraction with olivine host: The roles of competitor and spectator ions.

The lithium supply issue mainly lies in the inability of current mining methods to access lithium sources of dilute concentrations and complex chemistry. Electrochemical intercalation has emerged as a highly selective method for lithium extraction; however, limited source compositions have been studied, which is insufficient to predict its applicability to the wide range of unconventional water sources (UWS). This work addresses the feasibility and identifies the challenges of Li extraction by electrochemical intercalation from UWS, by answering three questions: 1) Is there enough Li in UWS? 2) How would the solution compositions affect the competition of Li+ to major ions (Na+/Mg2+/K+/Ca2+)? 3) Does the complex solution composition affect the electrode stability? Using one-dimensional olivine FePO4 as the model electrode, we show the complicated roles of major ions. Na+ acts as the competitor ion for host storage sites. The competition from Na+ grants Mg2+ and Ca2+ being only the spectator ions. However, Mg2+ and Ca2+ can significantly affect the charge transfer of Li+ and Na+, therefore affecting the Li selectivity. We point to improving the selectivity of Li+ to Na+ as the key challenge for broadening the minable UWS using the olivine host.

Cation Triggered Self-Assembly of α-Lactalbumin Nanotubes.

Metal ions play a dual role in biological systems. Although they actively participate in vital life processes, they may contribute to protein aggregation and misfolding and thus contribute to development of diseases and other pathologies. In nanofabrication, metal ions mediate the formation of nanostructures with diverse properties. Here, we investigated the self-assembly of α-lactalbumin into nanotubes induced by coordination with metal ions, screened among the series Mn2+, Co2+, Ni2+, Zn2+, Cd2+, and Au3+. Our results revealed that the affinity of metal ions toward hydrolyzed α-lactalbumin peptides not only impacts the kinetics of nanotube formation but also influences their length and rigidity. These findings expand our understanding of supramolecular assembly processes in protein-based materials and pave the way for designing novel materials such as metallogels in biochip and biosensor applications.

Polarity-Mediated Antisolvent Control Enables Efficient Lanthanide-Based near-Infrared Perovskite LEDs.

Alloying lanthanide ions (Yb3+) into perovskite quantum dots (Yb3+:CsPb(Cl1-xBrx)3) is an effective method to achieve efficient near-infrared (NIR) luminescence (>950 nm). Increasing the Yb3+ alloying ratio in the perovskite matrix enhances the luminescence intensity of Yb3+ emission at 990 nm. However, high Yb3+ alloying (>15%) results in vacancy-induced inferior material stability. In this work, we developed a polarity-mediated antisolvent manipulation strategy to resolve the incompatibility between a high Yb3+ alloying ratio and inferior stability of Yb3+:CsPb(Cl1-xBrx)3. Precise control of solution polarity enables increased uniformity of the perovskite matrix with fewer trap densities. Employing this strategy, we obtain Yb3+:CsPb(Cl1-xBrx)3 with the highest Yb3+ alloying ratio of 30.2% and a 2-fold higher electroluminescence intensity at 990 nm. We lever the engineered Yb3+:CsPb(Cl1-xBrx)3 to fabricate NIR-LEDs, achieving a peak external quantum efficiency (EQE) of 8.5% at 990 nm: this represents the highest among perovskite NIR-LEDs with an emission wavelength above 950 nm.

Cu(II) Specifically Disassembles Insulin Amyloid Nanostructures via Direct Interaction with Cross-ß Fibrils.

In this work, we demonstrate direct evidence of the antiamyloid potential of Cu(II) ions against amyloid formation of insulin. The Cu(II) ions were found to efficiently disassemble the preformed amyloid nanostructures into soluble species and suppress monomer fibrillation under aggregation-prone conditions. The direct interaction of Cu(II) ions with the cross-ß structure of amyloid fibrils causes substantial disruption of both the interchain and intrachain interactions, predominantly the H-bonds and hydrophobic contacts. Further, the Cu(II) ions show a strong affinity for the aggregation-prone conformers of the protein and inhibit their spontaneous self-assembly. These results reveal the possible molecular mechanism for the antiamyloidogenic potential of Cu(II) which could be important for the development of metal-ion specific therapeutic strategies against amyloid linked complications.

SpectiCal: m/z Calibration of MS2 Peptide Spectra Using Known Low Mass Ions.

Most tandem mass spectrometry fragmentation spectra have small calibration errors that can lead to suboptimal interpretation and annotation. We developed SpectiCal, a software tool that can read mzML files from data-dependent acquisition proteomics experiments in parallel, compute m/z calibrations for each file prior to identification analysis based on known low-mass ions, and produce information about frequently observed peaks and their explanations. Using calibration coefficients, the data can be corrected to generate new calibrated mzML files. SpectiCal was tested using five public data sets, creating a table of commonly observed low-mass ions and their identifications. Information about the calibration and individual peaks is written in PDF and TSV files. This includes information for each peak, such as the number of runs in which it appears, the percentage of spectra in which it appears, and a plot of the aggregated region surrounding each peak. SpectiCal can be used to compute MS run calibrations, examine MS runs for artifacts that might hinder downstream analysis, and generate tables of detected low-mass ions for further analysis. SpectiCal is freely available at https://github.com/PlantProteomes/SpectiCal.

Experimentally Determined Diagnostic Ions for Identification of Peptide Glycotopes.

The analysis of the structures of glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment on glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic analysis by mass spectrometry currently can provide significant, yet incomplete, information about the glycans present, including the glycan monosaccharide composition and in some circumstances the site(s) of glycosylation. Advancements in mass spectrometric resolution, using high-mass accuracy instrumentation and tailored MS/MS fragmentation parameters, coupled with a dedicated definition of diagnostic fragmentation ions have enabled the determination of some glycan structural features, or glycotopes, expressed on glycopeptides. Here we present a collation of diagnostic glycan fragments produced by traditional positive-ion-mode reversed-phase LC-ESI MS/MS proteomic workflows and describe the specific fragmentation energy settings required to identify specific glycotopes presented on N- or O-linked glycopeptides in a typical proteomics MS/MS experiment.

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11.

Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-ß-lactamase domain and a ß-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end processing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endonuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.

Analysis of potassium ion diffusion from neurons to capillaries: Effects of astrocyte endfeet geometry.

Neurovascular coupling (NVC) refers to a local increase in cerebral blood flow in response to increased neuronal activity. Mechanisms of communication between neurons and blood vessels remain unclear. Astrocyte endfeet almost completely cover cerebral capillaries, suggesting that astrocytes play a role in NVC by releasing vasoactive substances near capillaries. An alternative hypothesis is that direct diffusion through the extracellular space of potassium ions (K+ ) released by neurons contributes to NVC. Here, the goal is to determine whether astrocyte endfeet present a barrier to K+ diffusion from neurons to capillaries. Two simplified 2D geometries of extracellular space, clefts between endfeet, and perivascular space are used: (i) a source 1 µm from a capillary; (ii) a neuron 15 µm from a capillary. K+ release is modelled as a step increase in [K+ ] at the outer boundary of the extracellular space. The time-dependent diffusion equation is solved numerically. In the first geometry, perivascular [K+ ] approaches its final value within 0.05 s. Decreasing endfeet cleft width or increasing perivascular space width slows the rise in [K+ ]. In the second geometry, the increase in perivascular [K+ ] occurs within 0.5 s and is insensitive to changes in cleft width or perivascular space width. Predicted levels of perivascular [K+ ] are sufficient to cause vasodilation, and the rise time is within the time for flow increase in NVC. These results suggest that direct diffusion of K+ through the extracellular space is a possible NVC signalling mechanism.

From ferroptosis to cuproptosis, and calcicoptosis, to find more novel metals-mediated distinct form of regulated cell death.

Regulated cell death (RCD), also known as programmed cell death (PCD), plays a critical role in various biological processes, such as tissue injury/repair, development, and homeostasis. Dysregulation of RCD pathways can lead to the development of many human diseases, such as cancer, neurodegenerative disorders, and cardiovascular diseases. Maintaining proper metal ion homeostasis is critical for human health. However, imbalances in metal levels within cells can result in cytotoxicity and cell death, leading to a variety of diseases and health problems. In recent years, new types of metal overload-induced cell death have been identified, including ferroptosis, cuproptosis, and calcicoptosis. This has prompted us to examine the three defined metal-dependent cell death types, and discuss other metals-induced ferroptosis, cuproptosis, and disrupted Ca2+ homeostasis, as well as the roles of Zn2+ in metals' homeostasis and related RCD. We have reviewed the connection between metals-induced RCD and various diseases, as well as the underlying mechanisms. We believe that further research in this area will lead to the discovery of novel types of metal-dependent RCD, a better understanding of the underlying mechanisms, and the development of new therapeutic strategies for human diseases.