Protein Ions Research Articles

Investigating ion transport inside the pentameric ion channel encoded in COVID-19 E protein.

Ion flow inside an ion channel can be described through continuum based Born-Poisson-Nernst-Planck (BPNP) equations in conjunction with the Lennard-Jones potential. Keeping in mind the ongoing pandemic, in this study, an attempt has been made to understand the selectivity and the current voltage relation of the COVID-19 E protein pentameric ion channel. Two ionic species, namely Na^{+} and Cl^{-}, have been considered here. E protein is one of the smallest structural protein which is embedded in the outer membrane of the virus. Once the virus is inside the host cell, this protein is expressed abundantly and is responsible for activities such as replication and budding of the virus. In the literature, we can find a few experimental studies focusing on understanding the activity of the channel formed by E proteins of different viruses. Here, we attempt the same study for the COVID-19 E protein ion channel through mathematical modeling. The channel geometry is calculated from the protein data bank file which was provided by NARLabs, Taiwan, using the hole program. Further, it was used to obtain the charge distribution using the pdbtopqr online program. The immersed boundary-lattice Boltzmann method (IB-LBM) has been implemented to numerically solve the system of equations in the channel generated by the protein data bank file. Further, an in-house code which operates on multiple GPUs and uses the cuda platform has been developed to achieve the goal of performing the current investigation.

Ca2+-mediated enhancement of anesthetic diffusion across phospholipid multilamellar systems

Although sharing common properties with other divalent cations, calcium ions induce fine-tuned electrostatic effects essential in many biological processes. Not only related with protein structure or ion channels, calcium is also determinant for other biomolecules such as lipids or even drugs. Cellular membranes are the first interaction barriers for drugs. Depending on their hydrophilic, hydrophobic or amphipathic properties, they have to overcome such barriers to permeate and diffuse through inner lipid bilayers, cells or even tissues. In this context, the role of calcium in the permeation of cationic amphiphilic drugs (CADs) through lipid membranes is not well understood. We combine differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) to investigate the effect of Ca2+ on the interlamellar diffusion kinetics of the local anesthetic tetracaine (TTC) in multilamellar artificial membrane systems. Our DSC results show the interesting phenomenon that TTC diffusion can be modified in two different ways in the presence of Ca2+. Furthermore, TTC diffusion exhibits a thermal-dependent membrane interaction in the presence of Ca2+. The FTIR results suggest the presence of ion-dipole interactions between Ca2+ and the carbonyl group of TTC, leading us to hypothesize that Ca2+ destabilizes the hydration shell of TTC, which in turn diffuses deeper into the multilamellar lipid structures. Our results demonstrate the relevance of the Ca2+ ion in the drug permeation and diffusion through lipid bilayers.

Protein ion yield enhancement in matrix-assisted laser desorption/ionization mass spectrometry after sample and matrix low-pressure glow discharge plasma irradiation.

Plasma-assisted ionization is widely used in mass spectrometry; in this study, a low-pressure glow discharge is introduced as a new method to improve the detection of large proteins, and bovine serum albumin (BSA) is used as a protein model. The treatment of analyte, matrix, and the matrix/analyte mixture is evaluated under optimal conditions. Low-pressure radio-frequency capacitively coupled plasma (RF-CCP) treatment is utilized in the sample preparation step of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to enhance the protein MALDI ion signal. Plasma treatment can be an effective tool for enhancing the non-covalent binding of the analyte with the matrix, incorporation of the analyte into the matrix, production of matrix/analyte crystals, and analyte protonation through plasma activation, resulting in an improved MALDI ion signal. Fourier-transform infrared (FTIR) spectroscopy allows us to distinguish between the functional groups of plasma-treated and control samples. In addition, optical emission spectroscopy (OES) determines the plasma species, and zeta potential analysis characterizes the potential difference between plasma-treated and control samples before MALDI-TOF MS analysis. Plasma-treated BSA can provide a five-times enhancement of ion intensity. The combination of the plasma-treated analyte with the plasma-treated matrix leads to an increase in the ion intensity by a factor of 14. Low-pressure glow discharge plasma treatment greatly enhances MALDI ion signals, with a noticeable increase in incorporation, co-crystallization, protonation, and the concentration of the sample functional groups.

Enhanced Collision Induced Unfolding and Electron Capture Dissociation of Native-like Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.

Simultaneous Multiplexed Detection of Protein and Metal Ions by a Colorimetric Microfluidic Paper-based Analytical Device.

In order to improve the efficiency of disease diagnosis and environmental monitoring, it is desirable to detect the concentration of proteins and metal ions simultaneously, since the current popular diagnostic platform can only detect proteins or metal ions independently. In this work, we developed a colorimetric microfluidic paper-based analytical device (µPAD) for simultaneous determination of protein (bovine serum albumin, BSA) and metal ions [Fe(III) and Ni(II)]. The µPAD consisted of one central zone, ten reaction zones and ten detection zones in one device, in which reaction solutions were effectively optimized for different types of chromogenic reactions. Fe(III), Ni(II) and BSA can be easily identified by the colored products, and their concentrations are in good accordance with color depth based on the established standard curves. The detection limits are 0.1 mM for Fe(III), 0.5 mM for Ni(II) and 1µM for BSA, respectively. Best of all, we demonstrated the efficiency of the µPAD with accurate detection of Fe(III), Ni (II) and BSA from river water samples within 15 minutes. The µPAD detection is efficient, instrument-free, and easy-to-use, holding great potential for simultaneous detection of cross type analytes in numerous diagnostic fields.

Transmembrane Ion Channels Formed by a Star of David [2]Catenane and a Molecular Pentafoil Knot.

A (FeII)6-coordinated triply interlocked (“Star of David”) [2]catenane (612 link) and a (FeII)5-coordinated pentafoil (51) knot are found to selectively transport anions across phospholipid bilayers. Allostery, topology, and building block stoichiometry all play important roles in the efficacy of the ionophoric activity. Multiple FeII cation coordination by the interlocked molecules is crucial: the demetalated catenane exhibits no anion binding in solution nor any transmembrane ion transport properties. However, the topologically trivial, Lehn-type cyclic hexameric FeII helicates—which have similar anion binding affinities to the metalated Star of David catenane in solution—also display no ion transport properties. The unanticipated difference in behavior between the open- and closed-loop structures may arise from conformational restrictions in the linking groups that likely enhances the rigidity of the channel-forming topologically complex molecules. The (FeII)6-coordinated Star of David catenane, derived from a hexameric cyclic helicate, is 2 orders of magnitude more potent in terms of ion transport than the (FeII)5-coordinated pentafoil knot, derived from a cyclic pentamer of the same building block. The reduced efficacy is reminiscent of multisubunit protein ion channels assembled with incorrect monomer stoichiometries.

In silico identification of Tretinoin as a SARS-CoV-2 envelope (E) protein ion channel inhibitor

Viroporins are oligomeric, pore forming, viral proteins that play critical roles in the life cycle of pathogenic viruses. Viroporins like HIV-1 Vpu, Alphavirus 6 K, Influenza M2, HCV p7, and Picornavirus 2B, form discrete aqueous passageways which mediate ion and small molecule transport in infected cells. The alterations in host membrane structures induced by viroporins is essential for key steps in the virus life cycle like entry, replication and egress. Any disruption in viroporin functionality severely compromises viral pathogenesis. The envelope (E) protein encoded by coronaviruses is a viroporin with ion channel activity and has been shown to be crucial for the assembly and pathophysiology of coronaviruses. We used a combination of virtual database screening, molecular docking, all-atom molecular dynamics simulation and MM-PBSA analysis to test four FDA approved drugs - Tretinoin, Mefenamic Acid, Ondansetron and Artemether - as potential inhibitors of ion channels formed by SARS-CoV-2 E protein. Interaction and binding energy analysis showed that electrostatic interactions and polar solvation energy were the major driving forces for binding of the drugs, with Tretinoin being the most promising inhibitor. Tretinoin bound within the lumen of the channel formed by E protein, which is lined by hydrophobic residues like Phe, Val and Ala, indicating its potential for blocking the channel and inhibiting the viroporin functionality of E. In control simulations, tretinoin demonstrated a lower binding energy with a known target as compared to SARS-CoV-2 E protein. This work thus highlights the possibility of exploring Tretinoin as a potential SARS-CoV-2 E protein ion channel blocker and virus assembly inhibitor, which could be an important therapeutic strategy in the treatment for coronaviruses.

Responses by African catfish ( Clarias gariepinus ) fed diets containing fish visceral meal as fishmeal replacer

The responses (growth, economic performance and physiology) in African catfish (Clarias gariepinus) fed diets containing fish visceral meal as fishmeal replacer were investigated in a 56-day feeding trial. Five experimental diets, formulated using catfish visceral meal to replace fishmeal protein at 0%, 15%, 30%, 45% and 60%, were fed to triplicate fish group (n = 15 fish/replicate; 5.73 ± 0.05 g). The weight gained by fish fed control diet was significantly higher (p < .05) than fish fed test diets. However, no statistical variations (p > .05) existed in the survival, protein productive value, whole body composition, nutrient retention, liver total protein, sodium and calcium ion concentration. The hepatic metabolic (AST, ALT and ALP) and oxidative (SOD and CAT) enzymes activities and glutathione were not significantly different (p > .05) between fish fed test and control diets except fish fed D15T that had statistically higher (p < .05) values of ALT, SOD enzyme activities, glutathione, potassium and chloride ions. Economic performance of feeding diets containing catfish visceral meal to Clarias gariepinus showed that the incidence of cost was significantly lowered (p < .05) among catfish visceral meal fed group resulting into increase in profit index and profit per kg of catfish produced. However, statistically similarity (p > .05) exists with control group up to 30% replacement level.

Genome-wide discovery of OsHOX24-binding sites and regulation of desiccation stress response in rice.

OsHOX24 mediates regulation of desiccation stress response via complex regulatory network as indicated by its binding to several target genes including transcription factors in rice. HD-ZIP I subfamily of homeobox transcription factors (TFs) are involved in abiotic stress responses and plantdevelopment. Previously, we demonstrated the role of OsHOX24, a member of HD-ZIP I subfamily, in abiotic stress responses. In this study, we identified downstream targets of OsHOX24 under control and desiccation stress conditions via chromatin immunoprecipitation-sequencing (ChIP-seq) approach in wild-type and OsHOX24 over-expression transgenic in rice. OsHOX24-binding sites in each sample and differential binding sitesbetween the samples were detected at various genomic locations, including genic and intergenic regions. Gene ontology enrichment analysis revealed that OsHOX24 direct target genes were involved in several biological processes, including plant development, ABA-mediated signalling pathway, ubiquitin-dependent protein catabolic process, ion transport, abiotic and biotic stress responses besides transcriptional and translational regulation. The enrichment of several cis-regulatory motifs representing binding sites of other TFs, such as ABFs, ERF1, MYB1, LTREs and SORLIP2, suggested the involvement of OsHOX24 in a complex regulatory network. These findings indicate that OsHOX24-mediated desiccation stress regulation involves modulation of a plethora of target genes, which participate in diverse pathways in rice.

Protein kinase CK2 and ion channels (Review)

Protein kinase CK2 appears as a tetramer or higher molecular weight oligomer composed of catalytic CK2α, CK2α' subunits and non-catalytic regulatory CK2β subunits or as individual subunits. It is implicated in a variety of different regulatory processes, such as Akt signalling, splicing and DNA repair within eukaryotic cells. The present review evaluates the influence of CK2 on ion channels in the plasma membrane. CK2 phosphorylates platform proteins such as calmodulin and ankyrin G, which bind to channel proteins for a physiological transport to and positioning into the membrane. In addition, CK2 directly phosphorylates a variety of channel proteins directly to regulate opening and closing of the channels. Thus, modulation of CK2 activities by specific inhibitors, by siRNA technology or by CRISPR/Cas technology has an influence on intracellular ion concentrations and thereby on cellular signalling. The physiological regulation of the intracellular ion concentration is important for cell survival and correct intracellular signalling. Disturbance of this regulation results in a variety of different diseases including epilepsy, heart failure, cystic fibrosis and diabetes. Therefore, these effects should be considered when using CK2 inhibition as a treatment option for cancer.

Hydrophobic dewetting in gating and regulation of transmembrane protein ion channels.

Water is at the heart of almost all biological phenomena, without which no life that we know of would have been possible. It is a misleadingly complex liquid that exists in near coexistence with the vapor phase under ambient conditions. Confinement within a hydrophobic cavity can tip this balance enough to drive a cooperative dewetting transition. For a nanometer-scale pore, the dewetting transition leads to a stable dry state that is physically open but impermeable to ions. This phenomenon is often referred to as hydrophobic gating. Numerous transmembrane protein ion channels have now been observed to utilize hydrophobic gating in their activation and regulation. Here, we review recent theoretical, simulation, and experimental studies that together have started to establish the principles of hydrophobic gating and discuss how channels of various sizes, topologies, and biological functions can utilize these principles to control the thermodynamic properties of water within their interior pores for gating and regulation. Exciting opportunities remain in multiple areas, particularly on direct experimental detection of hydrophobic dewetting in biological channels and on understanding how the cell may control the hydrophobic gating in regulation of ion channels.

Polypeptide backbone cleavage on the C-terminal side of asparagine residues of metastable protein ions analyzed by MALDI-TOF-TOF-MS/MS and post-source decay

The gas phase dissociation of B-subunits of Shiga toxin 2 (Stx2) subtypes expressed by pathogenic Escherichia coli have been analyzed using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry and post-source decay (PSD). Two Stx2 subtypes (a and c) are distinguished by two amino acid substitutions involving aspartic acid (D), asparagine (N) and alanine (A) residues. In addition to polypeptide backbone cleavage (PBC) on the C-terminal side of D and glutamic acid (E) residues by the aspartic acid effect (AAE) fragmentation mechanism, PBC is also observed on the C-terminal side of N-residues. It is estimated that N-residues are at least an order-of-magnitude less efficient than D-residues at PBC. As the side-chains of D and N residues are of equal length, the difference in PBC efficiency is likely due to differences in the gas phase acidities (ΔΔHacid ≈ 0.4 eV) of their respective side-chain functional groups: [−(CO)OH] for D versus [−(CO)NH2] for N. It is proposed that the AAE mechanism for N-residues results in the formation of a b-type fragment with a 2,5-pyrrolidinedione (or succinimide) end-group at its C-terminus similar to previous proposals that predict formation of a b-type fragment with a 2,5-furanedione (or succinic anhydride) end-group for D-residues. As a D ↔ N substitution differs in mass by only 1 Da, the b-fragment would also differ by 1 Da (if there are no other residue substitutions) and the complementary y-type fragment would have exactly the same mass. In consequence, the relative abundance of fragment ions caused by a D versus N residue might be a better indicator as to which residue was responsible for PBC rather than relying solely upon fragment ion m/z for this determination.

Protonation isomers of highly charged protein ions can be separated in FAIMS-MS

High-field asymmetric waveform ion mobility spectrometry-mass spectrometry (FAIMS-MS) can resolve over an order of magnitude more conformers for a given protein ion than alternative methods. Such an expansion in separation space results, in part, from protein ions with masses of >29 kDa undergoing dipole alignment in the high electric field of FAIMS, and the resolution of ions that adopt pendular vs free rotor states. In this study, FAIMS-MS, collision-induced dissociation (CID), and travelling wave (TW) IMS-MS were used to investigate the pendular and free rotor states of protonated carbonic anhydrase II (CAII, 29 kDa). The electrospray ionization additive 1,2-butylene carbonate was used to increase protein charge states and ensure extended ion conformations were formed. For relatively high charge states in which dipole alignment occurs (30–38+), FAIMS-MS can baseline resolve the isobaric pendular and free rotor ion populations. For TWIMS-MS, these same charge states resulted in monomodal arrival time distributions with collision cross sections corresponding to highly extended ion conformations. Interestingly, CID of FAIMS-selected pendular and free rotor ion populations resulted in significantly different fragmentation patterns. For example, CID of the dipole aligned CAII 37+ resulted in cleavages C-terminal to residue 183, 192 and 196, whereas cleavage sites for the free rotor population occurred near residues 12 and 238. Given that the cleavage sites are ’directed’ by protonation sites in the CID of protein ions, and highly charged protein ions adopt extended conformations with the same or very similar collision cross sections, these results indicate that the pendular and free rotor populations separated in FAIMS can be attributed to protonation isomers. Moreover, the extent of protein ion charging in FAIMS-MS decreased substantially as the carrier gas flow rate decreased, indicating that ion charging in FAIMS-MS can be limited by proton-transfer reactions. Given that the total mass of proton charge carriers corresponds to less than 0.2% the mass of CAII, we anticipate that FAIMS-MS can be used to separate intact isobaric proteoforms with masses of at least ∼29 kDa that result from alternative sites of post-translational modifications.

Pre-clinical evaluation of metabolic and morphological changes in the body induced by 4-chlorobenzhydryl pip

Introduction. A significant medical and social challenge in occupational health poses a risk of chemical intoxication in the industrial synthesis of new drug compounds.The aim of the work was a pre-clinical study of metabolic and morphological changes in acute and subchronic exposure of 4-chlorobenzhydryl piperazine on the body, which will allow early diagnosis of health problems in workers in workshops where the reaction synthesis of piperazine compounds are used and carried out.Material and methods. A complex of toxic-hygienic and biochemical studies was carried out. The object of the research was 4-chlorobenzhydryl piperazine. Toxic properties were studied in experiments on white outbred rats. Single inhalation and subacute intragastric modes and methods of exposure were reproduced. Blood serum indices were analyzed: the activity of aspartate aminotransferase, alanine aminotransferase, catalase, the concentration of total protein, ceruloplasmin, urea, cholesterol, and glucose. The content of protein and chlorine ions was determined in the urine. Morphological studies were performed on the brain, heart, lungs, liver, kidneys, spleen, stomach, pancreas, and thyroid glands.Results. The changes in the biochemical indices of blood and urine in different ways of substance intake into the body were similar. An increase in the concentration of ceruloplasmin, which has a pronounced oxidase activity, was revealed. A decrease in catalase activity demonstrated the lack of antioxidant protection in the conditions of intoxication. A marker of dysfunctional liver disorders was an increase in the activity of aminotransferases. In rats, in the priming process, no changes in indices characterizing the functional state of the kidneys were revealed. 4-chlorobenzhydryl piperazine has the most pronounced toxic effect on the hepatobiliary system. Histological findings confirmed the development of mild toxic steatohepatosis in animals exposed to poisoning.Conclusion. When 4-chlorobenzhydryl piperazine is ingested, there is a risk of pathology, especially of the hepatobiliary system. The information content of biochemical indices that can become correct signs of health disorders in workers who are in contact with 4-chlorobenzhydryl piperazine is considered. The indices possessing not only high sensitivity but the activity of aminotransferases, the concentration of urea, and ceruloplasmin in blood serum also can provide diagnostically significant specificity.

Protein adsorption to (3-acrylamido propyl) trimethyl ammonium chloride-grafted Sepharose gel: Charge density reduction via copolymerizing with electroneutral monomer drastically increases uptake rate.

Polymer-grafting to porous materials is an effective way to create protein ion-exchangers of high capacity and uptake rate because the 3D architecture of the polymeric ion exchange groups provides high binding space and facilitated transport of the bound protein. Herein, a new anion exchanger was fabricated by grafting (3-acrylamide propyl) trimethyl ammonium chloride (APTAC) onto Sepharose FF gel for protein adsorption and chromatography. The ion exchanger, denoted as FF-pAPTAC, presented high capacity but limited uptake rate in bovine serum albumin (BSA) adsorption due to the high charge density of the cationic polymer chains. To solve the problem, we proposed to copolymerize APTAC with an electroneutral monomer, acrylamide (AM), onto Sepharose FF to modulate the charge density of the grafted polymer chains. By decreasing the feeding molar ratio of APTAC to AM, the ionic capacity (IC) of the copolymerized resins, FF-p(AM-APTAC)n (n denotes IC in mmol/L), decreased, but the chain length could be remained almost unchanged due to the similar reactivity ratios of the two monomers. With decreasing IC, the static adsorption capacity (qm) of FF-p(AM-APTAC)n decreased gradually because of the decline of protein binding sites. The uptake rate, however, represented by the ratio of effective pore diffusivity to the free solution diffusivity (De/D0), exhibited a strong uptrend with decreasing IC, reaching ~2.5-fold of the maximum observed with FF-pAPTACn. It is considered that the decrease of charge density weakened protein binding strength to the chains and increased the chain flexibility, which consequently facilitated the transport of bound proteins on the chains. Moreover, at the same IC, each FF-p(AM-APTAC)n displayed similar adsorption capacity but high uptake rate as compared with its FF-pAPTACn counterpart mainly due to the longer chain length of the copolymer. Particularly, ~6.5-fold enhancement of De/D0 was observed at IC=50±2mmol/L. Both high adsorption capacity and uptake rate made FF-p(AM-APTAC)n exhibit superior dynamic binding performance. The findings proved that reducing chain charge density by copolymerizing an electroneutral monomer was promising for fabrication of high-performance protein ion exchangers.

A label-free liquid crystal droplet-based sensor used to detect lead ions using single-stranded DNAzyme

A liquid crystal (LC) droplet pattern-based sensor used to detect lead ions (Pb2+) in phosphate buffer saline (PBS) solution using single-stranded DNAzyme has been reported. When Pb2+ were present, it bound to the DNAzyme, leading to the release of tetradecyltrimethylammonium bromide (OTAB). These OTAB molecules were absorbed onto the aqueous/LC interface and helped in arranging the LC orientation. A planar-to-homeotropic order transition of LC droplets was triggered during this event. The reported naked-eye optical sensor possesses a limit of detection (LOD) of about 0.7 nM in PBS and 0.8 nM in tap water. The proposed LC droplet-based sensor was highly sensitive and selective for label-free Pb2+ detection. This sensor also provided a platform for the detection of proteins, DNA, and other metal ions by modifying the receptor.

Increasing Collisional Activation of Protein Complexes Using Smaller Aperture Source Sampling Cones on a Synapt Q-IM-TOF Instrument with a Stepwave Source.

Quadrupole ion mobility time-of-flight (Q-IM-TOF) mass spectrometers have revolutionized investigation of native biomolecular complexes. High pressures in the sources of these instruments aid transmission of protein complexes through damping of kinetic energy by collisional cooling. As adducts are removed through collisional heating (declustering), excessive collisional cooling can prevent removal of nonspecific adducts from protein ions, leading to inaccurate mass measurements, broad mass spectral peaks, and obfuscation of ligand binding. We show that reducing the source pressure using smaller aperture source sampling cones (SC) in a Waters Synapt G2-Si instrument increases protein ion heating by decreasing collisional cooling, providing a simple way to enhance removal of adducted salts from soluble proteins (GroEL 14-mer) and detergents from a transmembrane protein complex (heptameric Staphylococcus aureus α-hemolysin, αHL). These experiments are supported by ion heating and cooling simulations which demonstrate reduced collisional cooling at lower source pressures. Using these easily swapped sample cones of different apertures is a facile approach to reproducibly extend the range of activation in Synapt-type instruments.

Formation of Gaseous Proteins via the Ion Evaporation Model (IEM) in Electrospray Mass Spectrometry.

The mechanisms whereby protein ions are released into the gas phase from charged droplets during electrospray ionization (ESI) continue to be controversial. Several pathways have been proposed. For native ESI the charged residue model (CRM) is favored; it entails the liberation of proteins via solvent evaporation to dryness. Unfolded proteins likely follow the chain ejection model (CEM), which involves the gradual expulsion of stretched-out chains from the droplet. According to the ion evaporation model (IEM) ions undergo electrostatically driven desorption from the droplet surface. The IEM is well supported for small precharged species such as Na+. However, it is unclear whether proteins can show IEM behavior as well. We examined this question using molecular dynamics (MD) simulations, mass spectrometry (MS), and ion mobility spectrometry (IMS) in positive ion mode. Ubiquitin was chosen as the model protein because of its structural stability which allows the protein charge in solution to be controlled via pH adjustment without changing the protein conformation. MD simulations on small ESI droplets (3 nm radius) showed CRM behavior regardless of the protein charge in solution. Surprisingly, many MD runs on larger droplets (5.5 nm radius) culminated in IEM ejection of ubiquitin, as long as the protein carried a sufficiently large positive solution charge. MD simulations predicted that nonspecific salt adducts are less prevalent for IEM-generated protein ions than for CRM products. This prediction was confirmed experimentally. Also, collision cross sections of MD structures were in good agreement with IMS data. Overall, this work reveals that the CRM, CEM, and IEM all represent viable pathways for generating gaseous protein ions during ESI. The IEM is favored for proteins that are tightly folded and highly charged in solution and for droplets in a suitable size regime.

90 YEARS OF PROGESTERONE: Steroid receptors as MAPK signaling sensors in breast cancer: let the fates decide.

Steroid hormone receptors (SRs) are classically defined as ligand-activated transcription factors that function as master regulators of gene programs important for a wide range of processes governing adult physiology, development, and cell or tissue homeostasis. A second function of SRs includes the ability to activate cytoplasmic signaling pathways. Estrogen (ER), androgen (AR), and progesterone (PR) receptors bind directly to membrane-associated signaling molecules including mitogenic protein kinases (i.e. c-SRC and AKT), G-proteins, and ion channels to mediate context-dependent actions via rapid activation of downstream signaling pathways. In addition to making direct contact with diverse signaling molecules, SRs are further fully integrated with signaling pathways by virtue of their N-terminal phosphorylation sites that act as regulatory hot-spots capable of sensing the signaling milieu. In particular, ER, AR, PR, and closely related glucocorticoid receptors (GR) share the property of accepting (i.e. sensing) ligand-independent phosphorylation events by proline-directed kinases in the MAPK and CDK families. These signaling inputs act as a 'second ligand' that dramatically impacts cell fate. In the face of drugs that reliably target SR ligand-binding domains to block uncontrolled cancer growth, ligand-independent post-translational modifications guide changes in cell fate that confer increased survival, EMT, migration/invasion, stemness properties, and therapy resistance of non-proliferating SR+ cancer cell subpopulations. The focus of this review is on MAPK pathways in the regulation of SR+ cancer cell fate. MAPK-dependent phosphorylation of PR (Ser294) and GR (Ser134) will primarily be discussed in light of the need to target changes in breast cancer cell fate as part of modernized combination therapies.

Small Molecule Channels Harness Membrane Potential to Concentrate Potassium in trk1Δtrk2Δ Yeast.

Many protein ion channels harness membrane potential to move ions in opposition to their chemical gradient. Deficiencies of such proteins cause several human diseases, including cystic fibrosis, Bartter Syndrome, and proximal renal tubular acidosis. Using yeast as a eukaryotic model system, we asked whether, in the context of a protein ion channel deficiency in vivo, small molecule channels could similarly harness membrane potential to concentrate ions. Trk potassium transporters use membrane potential to move potassium from a relatively low concentration outside cells (∼15 mM) to one of >10× higher inside (150-500 mM); trk1Δtrk2Δ are unable to concentrate potassium or grow in standard media. Here we show that potassium-permeable, but not potassium-selective, small-molecule ion channels formed by amphotericin B can harness membrane potential to concentrate potassium and thereby restore trk1Δtrk2Δ growth. This finding expands the list of potential human channelopathies that might be addressed by a molecular prosthetics approach.