Membrane Affinity Research Articles

Identification of a Potential Membrane-Targeting Sequence in the C-Terminus of the F Plasmid Segregation Protein SopA.

Stable maintenance and partitioning of the 'Fertility' plasmid or the F plasmid in its host Escherichia coli require the function of a ParA superfamily of proteins known as SopA. The mechanism by which SopA mediates plasmid segregation is well studied. SopA is a nucleoid-binding protein and binds DNA in an ATP-dependent but sequence non-specific manner. ATP hydrolysis stimulated by the binding of the SopBC complex mediates the release of SopA from the nucleoid. Cycles of ATP-binding and hydrolysis generate an ATPase gradient that moves the plasmid through a chemophoresis force. Nucleoid binding of SopA thus assumes a central role in its plasmid-partitioning function. However, earlier work also suggests that the F plasmid can be partitioned into anucleate cells, thus implicating nucleoid independent partitioning. Interestingly, SopA is also reported to be associated with the inner membrane of the bacteria. Here, we report the identification of a possible membrane-targeting sequence, a predicted amphipathic helix, at the C-terminus of SopA. Molecular dynamics simulations indicate that the predicted amphipathic helical motif of SopA has weak affinity for membranes. Moreover, we experimentally show that SopA can associate with bacterial membranes, is detectable in the membrane fractions of bacterial lysates, and is sensitive to the membrane potential. Further, unlike the wild-type SopA, a deletion of the C-terminal 29 amino acids results in the loss of F plasmids from bacterial cells.

Bioconcentration potential and microbial toxicity of onium cations in photoacid generators.

Despite the widespread utilization of onium salts as photoacid generators (PAGs) in semiconductor photolithography, their environmental, health, and safety (EHS) properties remain poorly understood. The present work reports the bioconcentration potential of five representative onium species (four sulfonium and one iodonium compound) by determining the octanol-water partition coefficient (POW) and lipid membrane affinity coefficient (KMA); microbial toxicity was evaluated using the bioluminescent bacterium Aliivibrio fischeri (Microtox bioassay). Four of the oniums exhibited varying degrees of hydrophobic (lipophilic) partitioning (log POW: 0.08-4.12; KMA: 1.70-5.62). A strong positive linear correlation was observed between log POW and KMA (KMA = log POW + 1.76, R2 = 0.99). The bioconcentration factors (log BCF) estimated from POW and KMA for the four oniums ranged from 0.13 to 3.67Lkg-1. Bis-(4-tert-butyl phenyl)-iodonium and triphenylsulfonium had 50% inhibitory concentrations (IC50) of 4.8 and 84.6μM, whereas the IC50 values of the other three oniums were not determined because these values were higher than their aqueous solubility. Given the increased regulatory scrutiny regarding the use and potential health impacts from onium PAGs, this study fulfills critical knowledge gaps concerning the EHS properties of PAG oniums, enabling more comprehensive evaluation of their environmental impacts and potential risk management strategies.

Membrane Binding of Antimicrobial Peptides Is Modulated by Lipid Charge Modification.

Peptide interactions with lipid bilayers play a key role in a range of biological processes and depend on electrostatic interactions between charged amino acids and lipid headgroups. Antimicrobial peptides (AMPs) initiate the killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defense mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysyl-phosphatidyl glycerol (Lys-PG). The resulting change in the membrane charge is suggested to reduce the binding of AMPs to membranes, thus impeding downstream AMP activity. Using coarse-grained molecular dynamics to investigate the effects of these modified lipids on AMP binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. More than 5000 simulations in total are used to define the relationship between lipid bilayer composition, peptide sequence (using five different membrane-active peptides), and peptide binding to membranes. The degree of interaction of a peptide with a membrane correlates with the membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that the lipid modifications due to MprF alter the energy barrier to peptide helix penetration of the bilayer. These results will offer a guide to the design of novel peptides, which addresses the issue of resistance via MprF-mediated membrane modification.

The Bionic Water Channel of Ultra-Short, High Affinity Carbon Nanotubes with High Water Permeability and Proton Selectivity

The development of the bionic water channel aims to replace the possible use of natural aquaporins (AQPs) for water purification, while retaining the ability of natural AQPs to carry out ultra-fast water transport and repel ions. Carbon nanotube channels (CNTCs) are a convenient membrane-based model system for studying nano-fluidic transport that replicates a number of key structural features of biological membrane channels. In this report, we describe protocols for CNTCs synthesis by ultrasound-assisted cutting of long CNTs in the presence of lipid amphiphiles. CNTCs have a similar thickness to the lipid membrane and high affinity for it. The ultra-short high-affinity CNTCs have high permeability and ion selectivity. The water permeability of the CNTCs is 1936 ± 123 μm/s, which is 2.3 times that of natural AQPs, and completely rejects salt ions. In general, carbon nanotubes represent a multifunctional nanopore building module for creating high-ranking functional bionic materials. This study has reference significance for the design of new bionic water channel and the actual development of bionic membrane based on CNTs.

Revealing the Mechanisms of Synergistic Action of Two Magainin Antimicrobial Peptides.

The study of peptide-lipid and peptide-peptide interactions as well as their topology and dynamics using biophysical and structural approaches have changed our view how antimicrobial peptides work and function. It has become obvious that both the peptides and the lipids arrange in soft supramolecular arrangements which are highly dynamic and able to change and mutually adapt their conformation, membrane penetration, and detailed morphology. This can occur on a local and a global level. This review focuses on cationic amphipathic peptides of the magainin family which were studied extensively by biophysical approaches. They are found intercalated at the membrane interface where they cause membrane thinning and ultimately lysis. Interestingly, mixtures of two of those peptides namely magainin 2 and PGLa which occur naturally as a cocktail in the frog skin exhibit synergistic enhancement of antimicrobial activities when investigated together in antimicrobial assays but also in biophysical experiments with model membranes. Detailed dose-response curves, presented here for the first time, show a cooperative behavior for the individual peptides which is much increased when PGLa and magainin are added as equimolar mixture. This has important consequences for their bacterial killing activities and resistance development. In membranes that carry unsaturations both peptides align parallel to the membrane surface where they have been shown to arrange into mesophases involving the peptides and the lipids. This supramolecular structuration comes along with much-increased membrane affinities for the peptide mixture. Because this synergism is most pronounced in membranes representing the bacterial lipid composition it can potentially be used to increase the therapeutic window of pharmaceutical formulations.

Mesoporous Silica Nanoparticles for Targeting Subcellular Organelles.

Current chemotherapy treatments lack great selectivity towards tumoral cells, which leads to nonspecific drug distribution and subsequent side effects. In this regard, the use of nanoparticles able to encapsulate and release therapeutic agents has attracted growing attention. In this sense, mesoporous silica nanoparticles (MSNs) have been widely employed as drug carriers owing to their exquisite physico-chemical properties. Because MSNs present a surface full of silanol groups, they can be easily functionalized to endow the nanoparticles with many different functionalities, including the introduction of moieties with affinity for the cell membrane or relevant compartments within the cell, thus increasing the efficacy of the treatments. This review manuscript will provide the state-of-the-art on MSNs functionalized for targeting subcellular compartments, focusing on the cytoplasm, the mitochondria, and the nucleus.

NH3 separation membranes with self-assembled gas highways induced by protic ionic liquids

• IL based hybrid membrane with NH 3 transport highways was developed. • Protic ILs induced self-assembled highways for enhanced NH 3 transport. • Protic ILs significantly improve NH 3 affinity and gas diffusion of membranes. • The NH 3 permeability and NH 3 /N 2 selectivity is simultaneously improved. As ecological pollution and resource waste caused by NH 3 emission become increasingly serious, thus the separation and recovery of NH 3 from exhausted gases is of great significance both for environment and economy. Herein, we present a kind of novel ionic liquids (ILs)-based hybrid membranes for efficient NH 3 separation. Protic ILs (PILs) are adopted to form self-assembled gas highways in the hybrid membranes with the nanostructures of a middle-sulfonated pentablock copolymer, exhibiting extremely high NH 3 affinity and gas diffusion. The highest NH 3 permeability and ideal NH 3 /N 2 selectivity is up to 2711 Barrer and 1407 at ambient temperature and 1 bar, much superior to the state-of-the-art membranes reported. The results indicated that the novel IL-based hybrid membranes are promising candidates for high-efficient NH 3 separation and purification.

CeO2 Nanomaterials from Diesel Engine Exhaust Induce DNA Damage and Oxidative Stress in Human and Rat Sperm In Vitro.

Cerium dioxide nanomaterials (CeO2 NMs) are widely used in nano-based diesel additives to decrease the emission of toxic compounds, but they have been shown to increase the emission of ultrafine particles as well as the amount of released Ce. The Organization for Economic Cooperation and Development included CeO2 NMs in the priority list of nanomaterials that require urgent evaluation, and the potential hazard of aged CeO2 NM exposure remains unexplored. Herein, human and rat sperm cells were exposed in vitro to a CeO2 NM-based diesel additive (called EnviroxTM), burned at 850 °C to mimic its release after combustion in a diesel engine. We demonstrated significant DNA damage after in vitro exposure to the lowest tested concentration (1 µg·L−1) using the alkaline comet assay (ACA). We also showed a significant increase in oxidative stress in human sperm after in vitro exposure to 1 µg·L−1 aged CeO2 NMs evaluated by the H2DCF-DA probe. Electron microscopy showed no internalization of aged CeO2 NMs in human sperm but an affinity for the head plasma membrane. The results obtained in this study provide some insight on the complex cellular mechanisms by which aged CeO2 NMs could exert in vitro biological effects on human spermatozoa and generate ROS.

Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation.

Extracellular vesicles (EV) have attracted much attention as potential biomarkers due to their protein, RNA and other nucleic acid content. The most common method used for EV isolation is differential ultracentrifugation (DU), however given the DU technical difficulties, other more practical methods have surged, such as membrane-affinity column commercial kits. Here, we assessed one commercial kit in terms of EV recovery and EV-derived RNA yield and compared it with a DU protocol. Our data shows that the commercial kit preparation results in a lower count of EV-like structures and a reduced expression of EV markers when compared to DU samples. Thus, apparently suggesting that the commercial kit had a lower EV yield. However, these findings did not reflect on RNA yield, which was greater with the commercial kit, even after an enzymatic treatment with proteinase K and RNAse A. We conclude that the kit has a higher EV-derived RNA yield in comparison to our DU protocol, suggesting that it may be the method of choice for RNA sequencing purposes.

Interaction of synthetic antimicrobial peptides of the Hylin a1 family with models of eukaryotic structures: Zwitterionic membranes and DNA

Antimicrobial peptides (AMPs) have been appointed as a possible alternative to traditional antibiotics in face of pathogens increasing resistance to conventional drugs. Hylin a1 (IFGAILPLALGALKNLIK), an AMP extracted from the skin secretion of a South American frog, Hypsiboas albopunctatus, was found to show a strong cytotoxicity against bacteria and fungus, but also a considerable hemolytic action. Considering the toxicity of the peptide in eukaryotic cells, this work focuses on investigating the effects of the interaction of the Hylin a1 analogues W6Hya1, D0W6Hya1 and K0W6Hya1 with models of eukaryotic structures, namely zwitterionic liposomes of dipalmitoyl phosphatidylcholine (DPPC) and calf-thymus DNA (CT DNA). Through intrinsic Trp fluorescence we determined that the peptide affinity for fluid DPPC bilayers follows the decreasing order: D0W6Hya1 (+2) > W6Hya1 (+3) » K0W6Hya1 (+4). Fluorescence data also indicate that the Trp residue in the more positively charged peptide, K0W6Hya1, is less deep in the bilayer than the residue in the other two peptides. This finding is supported by differential scanning calorimetry (DSC) data, which shows that both D0W6Hya1 and W6Hya1 disturb DPPC gel-fluid transition slightly more effectively than K0W6Hya1. DPPC DSC profiles are homogeneously disturbed by the three peptides, probably related to peptide-membrane diffusion. Surprisingly, the peptide that displays the lowest affinity for PC membranes and is located at the more superficial position in the bilayer, K0W6Hya1, is the most efficient in causing formation of pores on the membrane, as attested by carboxyfluorescein leakage assays. The three peptides were found to interact with CT DNA, with a deep penetration of the Trp residue into hydrophobic pockets of the double helix, as indicated by the significant blue shift on the Trp fluorescence, and the displacement of DNA-bound ethidium bromide by the peptides. The experiments of DNA electrophoresis confirm that Hylin peptides bind DNA in a concentration-dependent manner, inducing complete DNA retardation at the relative AMP/plasmid DNA weight ratio of ~17. These findings could help to better understand the AMPs toxic effects on eukaryotic cells, thus contributing to the design of healthier therapeutic agents.

Piezoelectric boron nitride nanosheets for high performance energy harvesting devices

The hexagonal boron nitride nanosheets (BNNSs) are one of the 2-dimensional(2D) materials, and have been explored for their applications in nanoelectronics etc, owing to their unique properties. Here, BNNSs are utilized to develop a high-performance hybrid piezo/triboelectric nanogenerator (PTEG). BNNSs of a few percentages in weight are incorporated into polydimethylsiloxane (PDMS) to form composites that are able to produce piezoelectric voltages up to ~ 5.4 V with d 33 ~ 12 pC/N. A PTEG composed of BNNS-PDMS and polyamide-6 (PA6) membranes ( 20 × 20 mm 2 ) demonstrates a current density of ~ 230 mA m − 2 , an output voltage of ~ 1870 V and a maximum power density of ~ 103.7 W m − 2 , more than three times higher than those of the control PDMS/PA6 nanogenerator. Electric polarization of the BNNS-PDMS membranes can further enhance the output of PTEGs. Detailed investigation reveals that the dramatically enhanced performance originates from the synergy of piezoelectric effect of BNNSs and increased electron affinity of BNNS-PDMS membranes, demonstrates the excellent potential of BNNS-based PTEGs. • We report a high-performance hybrid piezo/triboelectric nanogenerator utilizing few-layered hexagonal boron nitride nanosheets . • The BNNS-PDMS/PA6 PTEG demonstrates ultrahigh power density and stability with superior energy conversion efficiency. • The enhanced performance originates the synergy of the piezoelectric effects and the increased negative triboelectric properties of BNNS-PDMS composite membranes.

Application of peptides with an affinity for phospholipid membranes during the automated purification of extracellular vesicles

Extracellular vesicles (EVs), such as exosomes, have garnered increasing interest because of their potential clinical applications that range from diagnostics to therapeutics. The development of an automated and reproducible EV purification platform would therefore aid the introduction of EV biomarkers and therapies into the clinic. Here, we demonstrate that K8- as well as K-16 peptides (containing 8 and 16 lysine residues with dissociation constants of 102 nM and 11.6 nM for phosphatidylserine, respectively) immobilized on magnetic beads can capture small EVs (< 0.2 µm) from culture supernatants of MCF7 human breast cancer cells. Importantly, the bound EVs could be dissociated from the beads under mild conditions (e.g. 0.5 M NaCl), and the isolated EVs had the typical shapes of EVs under SEM and TEM with a mean particle size of 99 nm. Using the peptide-immobilized beads, we adapted a pre-existing bench top instrument for magnetic separation to perform automated EV purification with higher purity and yield than that obtained using the standard ultracentrifugation method.

Altered Conformation of α-Synuclein Drives Malfunction of Synaptic Vesicles in a Model of Parkinson's Disease

Misfolded, aggregated forms of alpha-synuclein (αSyn) are the principal protein component of Lewy bodies, the defining lesions of Parkinson’s disease (PD) and dementia with Lewy bodies. Increased copy number, missense mutations, and specific 5’ and 3’ regulatory elements of αSyn can all cause familial PD. Although αSyn participates in the regulation of synaptic vesicles, fundamental questions about the relationship between its structure and function in the presynaptic terminal are unanswered. Here, we systematically examined brain synaptosomes from non-transgenic (NTG) mice and transgenic mice expressing wild-type (WT) human αSyn or an amplified form of the E46K PD mutation (called 3K) that impairs the normal α-helical tetramerization of WT αSyn and increases its membrane affinity. Synaptosomes from transgenic mice expressing the 3K mutant had reduced Ca2+-dependent vesicle exocytosis, altered synaptic vesicle ultrastructure, impaired SNARE-complex assembly, and abnormal levels of select proteins involved in synaptic activity compared with NTG and WT human αSyn mice. We also developed a method to perform intra-synaptosomal NMR that revealed that WT αSyn participates in heterogeneous interactions with synaptic components. We show that these interactions depend on the presence of endogenous αSyn and that the 3K mutation diminishes the interplay between αSyn and the synapse. We conclude that the microenvironment inside presynaptic terminals is necessary for the proper conformation and interaction network of αSyn, where unfolded monomers of αSyn no longer represent the dominant conformation. We speculate that the inability to populate dynamically different conformational ensembles likely represents the early steps in αSyn misfunction that result in the neurotoxicity observed in PD and related synucleinopathies. With the advance of techniques such as cryo-electron tomography, in-cell single-molecule FRET, and in-cell NMR, in situ approaches like that presented here have the potential to address questions about the physiology of proteins such as αSyn that show diverse and changing conformations in their native, highly complex environments.

Abstract IA09: Control of PIP3 levels by PI3Kα and PTEN

Abstract Phosphatidylinositol 3,4,5-triphosphate (PIP3), a lipid second messenger, signals the initiation of cascades that result in, among other processes, cell proliferation, migration, and survival. PIP3 is generated by the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-3-kinase-α (PI3Kα) in response to activation by receptor tyrosine kinases (RTKs) or their substrates. PI3Kα has a residual constitutive activity that is counteracted by the action of PTEN, a phosphatase that is subjected to a large number of post-translational modifications. Thus, levels of PIP3 are controlled by a balance between the opposite actions of PI3Kα, a kinase, and PTEN, a phosphatase. Both enzymes are highly mutated in tumors. Oncogenic mutations have opposite effects on the activities of the two proteins: mutations activate PI3Kα but suppress the activity of PTEN. Both of these effects result in increased levels of PIP3 and provide selective advantage to the tumor cells carrying the mutations. In the physiologic activation of PI3Kα, the increase in activity is caused by the phosphotyrosine-containing region of the RTK dislodging the nSH2, a PI3Kα inhibitory domain from the regulatory subunit p85α, from its inhibitory interaction with the catalytic subunit p110α. A similar mechanism is used by some oncogenic mutations: they activate the enzyme by reducing the inhibitory action of the nSH2. Importantly, this effect is not a consequence of a transition to a new structure that can be identified as a new ground state. Instead, the mutations affect the dynamics of the protein in such a way that the new landscape favors catalytically competent conformations. Analysis of the interdomain interactions between the p110α and the p85α of the wild-type and the mutants suggests that the tumor-associated mutations effectively weaken the interactions between p110α and p85α by disrupting key stabilizing interactions. These findings support the notion that oncogenic mutations increase the enzymatic activity by enhancing the dynamics of the protein. Other oncogenic PI3Kα mutations increase the kinase activity by increasing the interaction of the enzyme with the membrane, and therefore augmenting accessibility to the substrate. In the case of PTEN, activity is controlled, at least in part, by the phosphorylation of four Ser/Thr residues (380, 382, 383, 385) in its 52-residue long C-terminal tail. This multiple phosphorylation results in an inhibition of the membrane-associated phosphatase activity. Using a variety of biochemical and biophysical techniques, we determined structural and physical chemical details of the mechanism controlling this inhibition. Briefly, in contrast to the unphosphorylated form, the tetra phosphorylated PTEN adopts a more compact conformation in which its carboxy terminal tail interacts with the C2 domain in a fashion that reduces its affinity for the cell membrane and diminishes its catalytic activity. Citation Format: Ignacia Echeverria, Yunlong Liu, Sweta Maheshwari, Mayukh Chakrabarti, Michelle Miller, David Bolduc, Philip Cole, Sandra B. Gabelli, L. Mario Amzel. Control of PIP3 levels by PI3Kα and PTEN [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA09.

Abstract PR08: AKT mutant allele-specific activation dictates pharmacologic sensitivities

Abstract The objective of this study is to explore the biochemical characteristics and therapeutic sensitivity profiles of novel, non-E17K, less frequent AKT1 mutations, and thereby expand the biomarker of sensitivity to AKT inhibition in molecularly defined cancer patients. AKT is a critical signaling node that translates phosphoinositide 3-kinase (PI3K) pathway stimulation into diverse cellular effects. Gain-of-function AKT1 mutations arise in diverse human cancers, of which E17K is the most common. Presence of AKT1 E17K mutation renders these tumors susceptible to AKT inhibition. Nevertheless, the long tail of potentially activating mutations in AKT is largely uncharacterized, thereby limiting our ability to act clinically in prospectively sequenced advanced cancer patients. We performed a population-scale candidate driver mutation discovery analysis in AKT1, AKT2 and AKT3 in a cohort of 41,075 retrospectively and prospectively sequenced primary and metastatic cancers and explored both their functional and biologic impact as well as their therapeutic sensitivity. Our results demonstrated that some, but not all, of the identified AKT missense mutations activated PI3K signaling in a growth factor-independent manner, and sensitized tumor cells to diverse AKT inhibitors. By contrast, we discovered a different class of small in-frame paralogous AKT duplication mutants that induced distinctive structural changes, leading to a far greater degree of membrane affinity, AKT activation, pathway dependence, and hypersensitivity to ATP-competitive AKT inhibitors, while conferring resistance to allosteric AKT inhibitors. Leveraging a co-clinical trial framework, we are now enrolling patients on the basis of these mutations in a basket study involving AKT alterations. One such case was that of a castration-resistant metastatic prostate cancer patient who harbored AKT2 duplication mutant, and subsequently responded to AKT inhibition. Collectively, our data indicate that the degree and mechanism of activation of oncogenic AKT mutants vary, thereby dictating allele-specific pharmacologic sensitivities to AKT inhibition. This abstract is also being presented as Poster B20. Citation Format: Tripti Shrestha Bhattarai, Tambudzai Shamu, Swati Patel, Alexander Gorelick, Matthew T. Chang, Elena I. Gavrila, JianJong Gao, Mark T.A. Donoghue, Paul S. Gao, Tara Soumerai, Wassim Abida, Lilian M. Smyth, David M. Hyman, David B. Solit, Barry S. Taylor. AKT mutant allele-specific activation dictates pharmacologic sensitivities [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR08.

Membrane dynamics in Leishmania amazonensis and antileishmanial activities of β-carboline derivatives

Two β-carboline compounds, 8i and 6d, demonstrated in vitro antileishmanial activity against Leishmania (L.) amazonensis promastigotes similar to that of miltefosine (MIL). Estimates of the membrane-water partition coefficient (KM/W) and the compound concentrations in the membrane (cm50) and aqueous phase (cw50) for half maximal inhibitory concentration were made. Whereas these biophysical parameters for 6d were not significantly different from those reported for MIL, 8i showed lower affinity for the parasite membrane (lower KM/W) and a lower concentration of the compound in the membrane required to inhibit the growth of the parasite (lower cm50). A 2-hour treatment of Leishmania promastigotes with the compounds 8i and 6d caused membrane rigidity in a concentration-dependent manner, as demonstrated by the electron paramagnetic resonance (EPR) technique and spin label method. This increased rigidity of the membrane was interpreted to be associated with the occurrence of cross-linking of oxidized cytoplasmic proteins to the parasite membrane skeleton. Importantly, the two β-carboline-oxazoline derivatives showed low hemolytic action, both in experiments with isolated red blood cells or with whole blood, denoting their great Leishmania/erythrocyte selectivity index. Using electron microscopy, changes in the membrane of both the amastigote and promastigote form of the parasite were confirmed, and it was demonstrated that compounds 8i and 6d decreased the number of amastigotes in infected murine macrophages. Furthermore, 8i and 6d were more toxic to the protozoa than to J774A.1 macrophages, with treated promastigotes exhibiting a decrease in cell volume, mitochondrial membrane potential depolarization, accumulation of lipid bodies, increased ROS production and changes in the cell cycle.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics.

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

Effect of Hydrophobicity on the Anticancer Activity of Fatty-Acyl-Conjugated CM4 in Breast Cancer Cells.

Antimicrobial peptides (AMPs) are important anticancer resources, and exploring AMP conjugates as highly effective and selective anticancer agents would represent new progress in cancer treatment. In this study, we synthesized C4–C16 fatty-acyl-conjugated AMP CM4 and investigated its physiochemical properties and cytotoxicity activity in breast cancer cells. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography (RP-HPLC) showed that long-chain fatty acyl (≥C12) conjugation prevented N-acyl-CM4 from trypsin hydrolysis. RP-HPLC and circular dichroism (CD) spectra showed that the hydrophobicity and helical content of N-acyl-CM4 increased with the acyl length. The acyl chain length was positively related to the cytotoxicity of C8–C16 conjugates, and C12–C16 fatty acyl conjugates exhibited significant cytotoxicity against MX-1, MCF-7, and MDA-MB-231 cells, with IC50 values <8 μM. Flow cytometry and confocal laser scanning microscopy results showed that N-acylated conjugation significantly increased the membrane affinity in breast cancer cells, and C12–C16 acyl conjugates were capable of translocating to the intracellular space, thereby targeting mitochondria and inducing apoptosis. N-acyl-CM4 showed low cytotoxicity against normal mammalian cells and erythrocytes, especially ≤C12 fatty acyl conjugates, exhibiting selective cytotoxicity to breast cancer cells. The current work indicated that increasing hydrophobicity by attaching long fatty acyl (≥C12) to AMPs may be an effective method to improve the anticancer activity, together with selectivity and resistance to trypsin hydrolysis. This finding provides a good strategy to develop AMPs as effective anticancer agents in the future.

AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase subunit, is critical for pollen tube growth in Arabidopsis

BackgroundGlycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8–2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8–2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8–2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized.ResultsHere we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8–2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG. Still, pigs-1 is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:GFP-PIGS transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings. However, the pPIGS:GFP-PIGS transgene seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:GFP-PIGS seedlings died soon after germination.ConclusionsCharacterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. Pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step.