Search Results (8)

Artificial peptides P4, A1 and A4 are homologous to amphipathic α-helical fragments of the influenza virus M1 protein. P4 and A4 contain the cholesterol recognition sequence CARC, which is absent in A1. As shown previously, P4 and A4 but not A1 have cytotoxic effects on some eukaryotic and bacterial cells. This might be caused by the dysfunction of cholesterol-dependent cellular structures, inhibition of the respiratory chain, or disruption of the membrane. Here, we analyzed the latter hypothesis by studying the uncoupling effect of the peptides on asolectin membranes. The influence of A4 on Δψ pre-formed either by the valinomycin-dependent K⁺ diffusion or by the activity of membrane-built cytochrome c oxidase (CcO) was studied on (proteo)liposomes. Also, we investigated the effect of P4, A1 and A4 on liposomes loaded with calcein. It is found that A4 in a submicromolar range causes an immediate and complete dissipation of diffusion Δψ across the liposomal membrane. Uncoupling of the CcO-containing proteoliposomes requires an order of magnitude of higher peptide concentration, which may indicate the sorption of A4 on the enzyme. The presence of cholesterol in the membrane significantly weakens the uncoupling. Submicromolar A4 and P4 cause the release of calcein from liposomes, indicating the formation of membrane pores. The process develops in minutes and is significantly decelerated by cholesterol. Micromolar A1 induces pore formation in a cholesterol-independent manner. We conclude that the peptides P4, A4 and, in higher concentrations, A1 form pores in the asolectin membrane. The CARC-mediated interaction of A4 and P4 with cholesterol impedes the peptide oligomerization necessary for pore formation. The rapid uncoupling effect of A4 is apparently caused by an increase in the proton conductivity of the membrane without pore formation. Full article

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca²⁺ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca²⁺ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca²⁺ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca²⁺ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca²⁺. The ion transport appears Ca²⁺-selective, since dispensing proteoliposomes in the presence of Mg²⁺ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca²⁺, thus confirming that the potential reduction was not only due to the binding of Ca²⁺ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca²⁺ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca²⁺ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles. Full article

Nanoencapsulation with proteoliposomes from natural membranes has been proposed as a carrier for the highly efficient delivery of mineral nutrients into plant tissues. Since Boron deficiency occurred frequently in crops, and is an element with low movement in tissues, in this work, nanoencapsulated B vs free B was applied to in vitro sweet potato plants to investigate the regulation of B transporters (aquaporins and specific transporters). Additionally, an metabolomic analysis was performed, and mineral nutrient and pigment concentrations were determined. The results showed high increases in B concentration in leaves when B was applied as encapsulated, but also Fe and Mn concentration increased. Likewise, the metabolomics study showed that single carbohydrates of these plants could be related to the energy need for increasing the expression of most NIP aquaporins (NIP1;2, NIP1;3; NIP4;1, NIP4;2, NIP5;1, NIP6;1, and NIP7) and boron transporters (BOR2, BOR4 and BOR7;1). Therefore, the results were associated with the higher mobility of encapsulated B into leaves and the stimulation of transport into cells, since after applying encapsulated B, the aforementioned NIPs and BORs increased in expression. Full article

The aquaporin-based biomimetic thin-film composite membrane (ABM-TFC) has demonstrated superior separation performance and achieved successful commercialization. The larger-scale production of the ABM membrane requires an appropriate balance between the performance and manufacturing cost. This study has systematically investigated the effects of proteoliposome concentration, protein-to-lipid ratio, as well as the additive on the separation performance of ABM for the purpose of finding the optimal preparation conditions for the ABM from the perspective of industrial production. Although increasing the proteoliposome concentration or protein-to-lipid ratio within a certain range could significantly enhance the water permeability of ABMs by increasing the loading of aquaporins in the selective layer, the enhancement effect was marginal or even compromised beyond an optimal point. Alternatively, adding cholesterol in the proteoliposome could further enhance the water flux of the ABM membrane, with minor effects on the salt rejection. The optimized ABM not only achieved a nearly doubled water flux with unchanged salt rejection compared to the control, but also demonstrated satisfactory filtration stability within a wide range of operation temperatures. This study provides a practical strategy for the optimization of ABM-TFC membranes to fit within the scheme of industrial-scale production. Full article

The mitochondrial 2-oxoglutarate carrier (OGC), isolated and purified from rat brain mitochondria, was reconstituted into proteoliposomes to study the interaction with hemin, a porphyrin derivative, which may result from the breakdown of heme-containing proteins and plays a key role in several metabolic pathways. By kinetic approaches, on the basis of the single binding centre gated pore mechanism, we analyzed the effect of hemin on the transport rate of OGC in uptake and efflux experiments in proteoliposomes reconstituted in the presence of the substrate 2-oxoglutarate. Overall, our experimental data fit the hypothesis that hemin operates a competitive inhibition in the 0.5–10 µM concentration range. As a consequence of the OGC inhibition, the malate/aspartate shuttle might be impaired, causing an alteration of mitochondrial function. Hence, considering that the metabolism of porphyrins implies both cytoplasmic and mitochondrial processes, OGC may participate in the regulation of porphyrin derivatives availability and the related metabolic pathways that depend on them (such as oxidative phosphorylation and apoptosis). For the sake of clarity, a simplified model based on induced-fit molecular docking supported the in vitro transport assays findings that hemin was as good as 2-oxoglutarate to bind the carrier by engaging specific ionic hydrogen bond interactions with a number of key residues known for participating in the similarly located mitochondrial carrier substrate binding site. Full article

Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGr^TM), a Mg²⁺-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg²⁺ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGr^TM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGr^TM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays. Full article

Many proteins are localized at the vacuolar membrane, but most of them are still poorly described, due to the inaccessibility of this membrane from the extracellular environment. This work focused on the characterization of the CAT2 transporter from S. lycopersicum (SlCAT2) that was previously overexpressed in E. coli and reconstituted in proteoliposomes for transport assay as [³H]Arg uptake. The orientation of the reconstituted transporter has been attempted and current data support the hypothesis that the protein is inserted in the liposome in the same orientation as in the vacuole. SlCAT2 activity was dependent on the pH, with an optimum at pH 7.5. SlCAT2 transport activity was stimulated by the increase of internal osmolality from 0 to 175 mOsmol while the activity was inhibited by the increase of external osmolality. K⁺, Na⁺, and Mg²⁺ present on the external side of proteoliposomes at physiological concentrations, inhibited the transport activity; differently, the cations had no effect when included in the internal proteoliposome compartment. This data highlighted an asymmetric regulation of SlCAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the SlCAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter GkApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of SlCAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes. Full article

We report the optimization of detergent-mediated reconstitution of an integral membrane-bound protein, full-length influenza M2 protein, by direct insertion into detergent-saturated liposomes. Detergent-mediated reconstitution is an important method for preparing proteoliposomes for studying membrane proteins, and must be optimized for each combination of protein and membrane constituents used. The purpose of the reconstitution was to prepare samples for site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) studies. Our goals in optimizing the protocol were to minimize the amount of detergent used, reduce overall proteoliposome preparation time, and confirm the removal of all detergent. The liposomes were comprised of (1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and the detergent octylglucoside (OG) was used for reconstitution. Rigorous physical characterization was applied to optimize each step of the reconstitution process. We used dynamic light scattering (DLS) to determine the amount of OG needed to saturate the preformed liposomes. During detergent removal by absorption with Bio-Beads, we quantified the detergent concentration by means of a colorimetric assay, thereby determining the number of Bio-Bead additions needed to remove all detergent from the final proteoliposomes. We found that the overnight Bio-Bead incubation used in previously published protocols can be omitted, reducing the time needed for reconstitution. We also monitored the size distribution of the proteoliposomes with DLS, confirming that the size distribution remains essentially constant throughout the reconstitution process. Full article