Bid induces cytochrome c-impermeable Bax channels in liposomes

Bax is a proapoptotic member of the Bcl-2 family of proteins. The Bax protein is dormant in the cytosol of normal cells and is activated upon induction of apoptosis. In apoptotic cells, Bax gets translocated to mitochondria, inserts into the outer membrane, oligomerizes and triggers the release of cytochrome c, possibly by channel formation. The BH3 domain-only protein Bid induces a conformational change in Bax before its insertion into the outer membrane. The mechanism by which Bid promotes Bax activation is not understood, and whether Bid is the only protein required for Bax activation is unclear. Here we report that recombinant full-length Bax (BaxFL) does not form channels in lipid bilayers when purified as a monomer. In contrast, in the presence of Bid cut with caspase 8 (cut Bid), Bax forms ionic channels in liposomes and planar bilayers. This channel-forming activity requires an interaction between cut Bid and Bax, and is inhibited by Bcl-xL. Moreover, in the absence of the putative transmembrane C-terminal domain, Bax does not form ionic channels in the presence of cut Bid. Cut Bid does not induce Bax oligomerization in liposomes and the Bax channels formed in the presence of cut Bid are not large enough to permeabilize vesicles to cytochrome c. In conclusion, our results suggest that monomeric BaxFL can form channels only in the presence of cut Bid. Cut Bid by itself is unable to induce Bax oligomerization in lipid membranes. It is suggested that another factor that might be present in mitochondria is required for Bax oligomerization.

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Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins

International Journal of Molecular Sciences ◽

10.3390/ijms21155496 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5496

Author(s):

Claire Siebert ◽

Corinne Mercier ◽

Donald K. Martin ◽

Patricia Renesto ◽

Beatrice Schaack

Keyword(s):

Outer Membrane ◽

Lipid Bilayers ◽

Lipid Membranes ◽

Iron Transport ◽

Molecular Mechanisms ◽

Protein A ◽

Membrane Vesicles ◽

Disodium Salt ◽

Outer Membrane Vesicles ◽

Novel Therapeutic Strategies

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.

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Reconstitution of the complement channel into lipid vesicles and planar bilayers starting from the fluid phase complex

Bioscience Reports ◽

10.1007/bf01117059 ◽

1985 ◽

Vol 5 (2) ◽

pp. 129-136 ◽

Cited By ~ 7

Author(s):

Gianfranco Menestrina ◽

Flavia Pasquali

Keyword(s):

Lipid Bilayers ◽

Membrane Attack Complex ◽

Fluid Phase ◽

Lipid Vesicles ◽

Ionic Channels ◽

Single Channels ◽

Planar Bilayers ◽

Low Concentrations ◽

Host Membrane ◽

Phase Complex

Proteolysis of the fluid phase complement complex SC5b-9 transforms it into an arnphiphilic molecule which resembles the membrane attack complex of complement and reconstitutes into lipid vesicles. Complement-containing vesicles prepared in this way can be made to fuse with planar lipid bilayers transferring their protein content to the host membrane. Massive conductance increases can thus be observed, which are due to the insertion of a large number of ionic channels into the membrane. Using low concentrations of vesicles, single channels can be studied.

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Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.12.4876-4883.2005 ◽

2005 ◽

Vol 49 (12) ◽

pp. 4876-4883 ◽

Cited By ~ 73

Author(s):

Axel Siroy ◽

Virginie Molle ◽

Christelle Lemaître-Guillier ◽

David Vallenet ◽

Martine Pestel-Caron ◽

...

Keyword(s):

Membrane Protein ◽

Acinetobacter Baumannii ◽

Outer Membrane ◽

Lipid Bilayers ◽

Outer Membrane Protein ◽

Carbapenem Resistance ◽

Protein Band ◽

Multidrug Resistant ◽

Channel Formation ◽

Artificial Lipid Bilayers

ABSTRACT It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (M w and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels.

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Non-linear Conductance, Rectification, and Mechanosensitive Channel Formation of Lipid Membranes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.592520 ◽

2021 ◽

Vol 8 ◽

Author(s):

Karis Amata Zecchi ◽

Thomas Heimburg

Keyword(s):

Lipid Bilayers ◽

Lipid Membranes ◽

Lipid Membrane ◽

Electrical Response ◽

Spontaneous Curvature ◽

Pressure Gradients ◽

Channel Formation ◽

Voltage Gated ◽

Non Linear ◽

Different Depths

There is mounting evidence that lipid bilayers display conductive properties. However, when interpreting the electrical response of biological membranes to voltage changes, they are commonly considered as inert insulators. Lipid bilayers under voltage-clamp conditions display current traces with discrete conduction-steps, which are indistinguishable from those attributed to the presence of protein channels. In current-voltage (I-V) plots they may also display outward rectification, i.e., voltage-gating. Surprisingly, this has even been observed in chemically symmetric lipid bilayers. Here, we investigate this phenomenon using a theoretical framework that models the electrostrictive effect of voltage on lipid membranes in the presence of a spontaneous polarization, which can be recognized by a voltage offset in electrical measurements. It can arise from an asymmetry of the membrane, for example from a non-zero spontaneous curvature of the membrane. This curvature can be caused by voltage via the flexoelectric effect, or by hydrostatic pressure differences across the membrane. Here, we describe I-V relations for lipid membranes formed at the tip of patch pipettes situated close to an aqueous surface. We measured at different depths relative to air/water surface, resulting in different pressure gradients across the membrane. Both linear and non-linear I-V profiles were observed. Non-linear conduction consistently takes the form of outward rectified currents. We explain the conductance properties by two mechanisms: One leak current with constant conductance without pores, and a second process that is due to voltage-gated pore opening correlating with the appearance of channel-like conduction steps. In some instances, these non-linear I-V relations display a voltage regime in which dI/dV is negative. This has also been previously observed in the presence of sodium channels. Experiments at different depths reveal channel formation that depends on pressure gradients. Therefore, we find that the channels in the lipid membrane are both voltage-gated and mechanosensitive. We also report measurements on black lipid membranes that also display rectification. In contrast to the patch experiments they are always symmetric and do not display a voltage offset.

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Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076640 ◽

1983 ◽

Vol 41 ◽

pp. 608-609

Author(s):

Neng-Bo He ◽

S.W. Hui

Keyword(s):

Lipid Bilayers ◽

Erythrocyte Membrane ◽

Lipid Membranes ◽

Surface Film ◽

Biological Membranes ◽

Electron Microscopic Observation ◽

Electron Microscopic ◽

Black Lipid Membranes ◽

Fine Mesh ◽

Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

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Cardiolipin-Containing Lipid Membranes Attract the Bacterial Cell Division Protein DivIVA

International Journal of Molecular Sciences ◽

10.3390/ijms22158350 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8350

Author(s):

Naďa Labajová ◽

Natalia Baranova ◽

Miroslav Jurásek ◽

Robert Vácha ◽

Martin Loose ◽

...

Keyword(s):

Cell Division ◽

Lipid Bilayers ◽

Bacterial Cell ◽

Lipid Membranes ◽

Model Organism ◽

Active Role ◽

Membrane Curvature ◽

Bacterial Cell Division ◽

Division Site ◽

Clostridioides Difficile

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.

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Effects of cytochrome c on the mitochondrial apoptosis-induced channel MAC

AJP Cell Physiology ◽

10.1152/ajpcell.00183.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. C1109-C1117 ◽

Cited By ~ 59

Author(s):

Liang Guo ◽

Dawn Pietkiewicz ◽

Evgeny V. Pavlov ◽

Sergey M. Grigoriev ◽

John J. Kasianowicz ◽

...

Keyword(s):

Cytochrome C ◽

Outer Membrane ◽

Cell Types ◽

Rnase A ◽

Mitochondrial Outer Membrane ◽

Dependent Manner ◽

Mitochondrial Apoptosis ◽

Noise Type ◽

Voltage Dependent

Recent studies indicate that cytochrome c is released early in apoptosis without loss of integrity of the mitochondrial outer membrane in some cell types. The high-conductance mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane early in apoptosis of FL5.12 cells. Physiological (micromolar) levels of cytochrome c alter MAC activity, and these effects are referred to as types 1 and 2. Type 1 effects are consistent with a partitioning of cytochrome c into the pore of MAC and include a modest decrease in conductance that is dose and voltage dependent, reversible, and has an increase in noise. Type 2 effects may correspond to “plugging” of the pore or destabilization of the open state. Type 2 effects are a dose-dependent, voltage-independent, and irreversible decrease in conductance. MAC is a heterogeneous channel with variable conductance. Cytochrome c affects MAC in a pore size-dependent manner, with maximal effects of cytochrome c on MAC with conductance of 1.9–5.4 nS. The effects of cytochrome c, RNase A, and high salt on MAC indicate that size, rather than charge, is crucial. The effects of dextran molecules of various sizes indicate that the pore diameter of MAC is slightly larger than that of 17-kDa dextran, which should be sufficient to allow the passage of 12-kDa cytochrome c. These findings are consistent with the notion that MAC is the pore through which cytochrome c is released from mitochondria during apoptosis.

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Long chain sphingomyelin depletes cholesterol from the cytoplasmic leaflet in asymmetric lipid membranes

RSC Advances ◽

10.1039/d1ra01464a ◽

2021 ◽

Vol 11 (37) ◽

pp. 22677-22682

Author(s):

Maria Lyngby Karlsen ◽

Dennis S. Bruhn ◽

Weria Pezeshkian ◽

Himanshu Khandelia

Keyword(s):

Outer Membrane ◽

Lipid Membranes ◽

Acyl Chain ◽

Outer Membrane Leaflet ◽

Long Chain

Long acyl chain sphingomyelin and saturated phospholipid tails in the outer membrane leaflet deplete cholesterol from the inner leaflet in mammalian membranes.

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The Use of Tethered Bilayer Lipid Membranes to Identify the Mechanisms of Antimicrobial Peptide Interactions with Lipid Bilayers

Antibiotics ◽

10.3390/antibiotics8010012 ◽

2019 ◽

Vol 8 (1) ◽

pp. 12 ◽

Cited By ~ 13

Author(s):

Amani Alghalayini ◽

Alvaro Garcia ◽

Thomas Berry ◽

Charles Cranfield

Keyword(s):

New Zealand ◽

Patch Clamp ◽

Lipid Bilayers ◽

Lipid Membranes ◽

Antimicrobial Agents ◽

Bilayer Lipid Membranes ◽

Bilayer Lipid ◽

Black Lipid Membranes ◽

Peptide Interactions ◽

New Research

This review identifies the ways in which tethered bilayer lipid membranes (tBLMs) can be used for the identification of the actions of antimicrobials against lipid bilayers. Much of the new research in this area has originated, or included researchers from, the southern hemisphere, Australia and New Zealand in particular. More and more, tBLMs are replacing liposome release assays, black lipid membranes and patch-clamp electrophysiological techniques because they use fewer reagents, are able to obtain results far more quickly and can provide a uniformity of responses with fewer artefacts. In this work, we describe how tBLM technology can and has been used to identify the actions of numerous antimicrobial agents.

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