Induce Pore Formation Research Articles

Quantifying the role of microstreaming in sonoporation

Cavitation microstreaming has been identified as an important mechanism underpinning sonoporation. The reported evidence includes experimental observation of the permeabilization of non-adherent cells or vesicles in microstreaming and theoretical and experimental estimates of the fluid shear stress required to induce pore formation in cell membranes and/or the activation of membrane components such as ion channels. The aim of this study was to assess the relative contribution of microstreaming to sonoporation observed in vitro with adherent cells, as commonly used in drug delivery studies. Streak velocimetry measurements were performed within a microfluidic cell of the microstreaming around a contrast agent microbubble driven at 0.5 and 1MHz and peak negative pressures up to 1MPa. The results suggest that, for the cell densities and microbubble concentrations typically reported in sonoporation studies, only a very small proportion of cells within a typical culture device would be exposed to sufficiently high shear stresses to be permeabilised. This finding is inconsistent with reported results for drug delivery efficiency and suggests that microstreaming may not be the dominant mechanism of sonoporation in this type of experiment.

Isolation, purification, identification, and discovery of the antibacterial mechanism of ld-phenyllactic acid produced by Lactiplantibacillus plantarum CXG9 isolated from a traditional Chinese fermented vegetable

Phenyllactic acid (PLA) is an antimicrobial compound and has been rarely reported to naturally occur in food sources with the exception of honey, sourdough, and some pickles. In this study, a traditional Chinese fermented vegetable, Stinky xiancaigeng, was found to contain high concentrations of PLA. A high PLA level-producing Lactiplantibacillus plantarum (L. plantarum) CXG9 was subsequently isolated and subjected to genomic analysis. Six L- and two d-lactate dehydrogenase genes were predicted, indicating that PLA produced by L. plantarum CXG9 contained L- and D-isomers. The ratio of L- and D-isomers was 11:1 by chiral high performance liquid chromatography analysis. LD-PLA from L. plantarum CXG9 cultures were then purified by a three-step procedure and identified by mass spectrometry. LD-PLA exhibited strong anti-Listeria monocytogenes activity, with a minimum inhibitory concentration of 2.4 mg/mL. The results of anti-Listeria monocytogenes mechanism demonstrated that LD-PLA damaged cell membrane integrity, increased membrane permeability, induced pore formation, and triggered intracellular material leakage by interacting with cell membrane proteins. These results presented here contribute to the use of PLA in further development and application as an antibacterial agent in the food industry.

Dynorphin A induces membrane permeabilization by formation of proteolipidic pores. Insights from electrophysiology and computational simulations

Dynorphins are endogenous neuropeptides that function as ligands for the κ-opioid receptor. In addition to opioid activity, dynorphins can induce several pathological effects such as neurological dysfunctions and cell death. Previous studies have suggested that Dynorphin A (DynA) mediates some pathogenic actions through formation of transient pores in lipid domains of the plasma membrane. Here, we use planar bilayer electrophysiology to show that DynA induces pore formation in negatively charged membranes.

What different physical techniques can disclose about disruptions on membrane structure caused by the antimicrobial peptide Hylin a1 and a more positively charged analogue

The present work monitors structural changes in anionic membranes (DPPG; 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) caused by the native antimicrobial peptide (AMP) Hylin a1 (Hya1; IFGAILPLALGALKNLIK-NH2) and its synthetic analogue K0Hya1 (KIFGAILPLALGALKNLIK-NH2), with an extra positive residue of lysine at the N-terminus of the peptide chain. Anionic membranes were used to mimic anionic lipids in bacteria membranes. Differential scanning calorimetry (DSC) evinced that both peptides strongly disrupt the lipid bilayers. However, whereas the native peptide (+3) induces a space-average and/or time-average disruption on DPPG bilayers, the more charged, K0Hya1 (+4), appears to be strongly attached to the membrane, clearly giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disrupted. The membrane fluorescent probe Laurdan indicates that, in average, the peptides increase the bilayer packing of fluid DPPG (above the lipid gel-fluid transition temperature) and/or decrease its polarity. Spin labels, incorporated into DPPG membrane, confirm, and extend the results obtained with Laurdan, indicating that the peptides increase the lipid packing both in gel and fluid DPPG bilayers. Therefore, our results confirm that Laurdan is often unable to monitor structural modifications induced on gel membranes by exogenous molecules. Through the measurement of the leakage of entrapped carboxyfluorescein (CF), a fluorescent dye, in DPPG large unilamellar vesicles it was possible to show that both peptides induce pore formation in DPPG bilayers. Furthermore, CF experiments show that Hylin peptides are strongly bound to DPPG bilayers in the gel phase, not being able to migrate to other DPPG vesicles. Here we discuss the complementarity of different techniques in monitoring structural alterations caused on lipid bilayers by Hylin peptides, and how it could be used to help in the understanding of the action of other exogenous molecules on biological membranes.

Porosity Formation in Thin Welded Joints of Al-MG-LI Alloys.

This work is about the study of the correlation of pore formation in welded joints of Al–MG–LI alloy with zirconium additives with the state of the base metal, thermal vacuum treatment, and welding technologies MIG and EBW. Metallographic analysis has been carried out, the phase composition of the alloy and weld metal has been investigated, and thermal cycles of welding have been calculated, allowing to estimate the residence time of metal in the alloying zone and weld metal in the liquid state. The nature of the allocation of strengthening fine-dispersed phases in the welded joints of the alloy has been determined. The regularity and character of pore formation in welded joints depending on the applicable thermal vacuum treatment (TVT) and welding technology have been revealed. It was established that TVT with subsequent hardening and aging has no effect on the phase composition of the alloy. However, this type of treatment contributes to the formation of a more homogeneous and uniform nature of the separation of fine-dispersed strengthening phases. It was revealed that the MIG technology (metal with and without TVT) is characterized by a large length of the fusion zone, the high residence time of metal in the fusion zone and weld metal in the liquid state, and the formation of pores. Phase formation in the temperature range of the beginning and end of the alloy crystallization occurs not only in the weld at the final stage of crystallization but also in the fusion zone, which may induce pore formation, whereas EBW welding shows the opposite trend and no pores. It was found that EBW technology prevents pore formation and makes it possible to obtain welded joints of 1420 Al alloys of the required quality.

Claudin-Targeted Suicide Gene Therapy for Claudin-Overexpressing Tumor Cells by Using Modified Clostridium perfringens Enterotoxin (CPE).

Bacterial toxins gain growing attention as potential cancer treatment due to their potent cytotoxic effects. Among the very different toxins with diverse modes of action, the Clostridium perfringens enterotoxin (CPE) is in focus to treat solid cancers. This toxin targets the tight junction proteins claudin-3 and -4 (Cldn-3/4), which are frequently overexpressed in solid cancers. Binding to these claudins induces pore formation in the host cell plasma membrane leading to rapid oncoleaking cell death of tumor cells. Based on this, extending the targeting of CPE beyond Cldn-3/4 is of interest, since other claudins, such as claudin-1 or -5 are often overexpressed in various cancer entities such as non-small-cell lung cancer (NSCLC) or papillary thyroid carcinoma. In this chapter we describe the modification of a CPE-encoding vector by structure-directed mutagenesis to either preferentially target Cldn-1 and -5 or to expand targeting to Cldn1-9 for improved broadened cytotoxic targeting of claudin-overexpressing tumors such as but not limited to lung cancer via CPE gene transfer.

Dynorphin A induces membrane permeabilization by formation of proteolipidic pores. Insights from electrophysiology and computational simulations

Dynorphins are endogenous neuropeptides that function as ligands for the κ-opioid receptor. In addition to opioid activity, dynorphins can induce several pathological effects such as neurological dysfunctions and cell death. Previous studies have suggested that Dynorphin A (DynA) mediates some pathogenic actions through formation of transient pores in lipid domains of the plasma membrane. Here, we use planar bilayer electrophysiology to show that DynA induces pore formation in negatively charged membranes. We find a large variability in pore conformations showing equilibrium conductance fluctuations, what disregards electroporation as the dominant mechanism of pore formation. Ion selectivity measurements showing cationic selectivity indicate that positive protein charges of DynA are stabilized by phosphatidyl serine negative charges in the formation of combined structures. We complement our study with computational simulations that assess the stability of diverse peptide arrangements in the hydrophobic core of the bilayer. We show that DynA is capable of assembling in charged membranes to form water-filled pores that conduct ions.

Disulfiram-loaded lactoferrin nanoparticles for treating inflammatory diseases.

Sepsis is a dysregulated immune response to infection and potentially leads to life-threatening organ dysfunction, which is often seen in serious Covid-19 patients. Disulfiram (DSF), an old drug that has been used to treat alcohol addiction for decades, has recently been identified as a potent inhibitor of the gasdermin D (GSDMD)-induced pore formation that causes pyroptosis and inflammatory cytokine release. Therefore, DSF represents a promising therapeutic for the treatment of inflammatory disorders. Lactoferrin (LF) is a multifunctional glycoprotein with potent antibacterial and anti-inflammatory activities that acts by neutralizing circulating endotoxins and activating cellular responses. In addition, LF has been well exploited as a drug nanocarrier and targeting ligands. In this study, we developed a DSF-LF nanoparticulate system (DSF-LF NP) for combining the immunosuppressive activities of both DSF and LF. DSF-LF NPs could effectively block pyroptosis and inflammatory cytokine release from macrophages. Treatment with DSF-LF NPs showed remarkable therapeutic effects on lipopolysaccharide (LPS)-induced sepsis. In addition, this therapeutic strategy was also applied to treat ulcerative colitis (UC), and substantial treatment efficacy was achieved in a murine colitis model. The underlying mode of action of these DSF-LF-NPs may contribute to efficiently suppressing macrophage-mediated inflammatory responses and ameliorating the complications caused by sepsis and UC. As macrophage pyroptosis plays a pivotal role in inflammation, this safe and effective biomimetic nanomedicine may offer a versatile therapeutic strategy for treating various inflammatory diseases by repurposing DSF.

Discovery of bilirubin as novel P2X7R antagonist with anti-tumor activity

As a unique ligand gated ion channel in the P2-receptor family, P2X7R is highly expressed in various tumors. The activated P2X7R facilitates tumor growth and metastasis. Hypoxia, inflammation and necrosis in the tumor microenvironment (TME) cause a large amount of adenosine triphosphate (ATP) accumulated in the TME. High concentration of ATP can abnormally activate P2X7R, which induces pore formation and further facilitates the Ca2+ ion influx and non-specific substance intake. Therefore, inhibition of P2X7R activation can be applied as a potential anti-tumor therapy strategy. However, there is currently no FDA approved drugs for this target for anti-tumor treatment. In this study, we identified bilirubin as novel P2X7R antagonist by using structure based virtual screening combined with cell based assays. Molecular docking studies indicated that bilirubin probably interacted with P2X7R by forming hydrogen-π interactions with residues V173, E174 and K311. The compound bilirubin inhibited the P2X7R gated EB intake by cancer cells. Meanwhile, bilirubin was capable to inhibit the cell proliferation and migration of P2X7R expressed HT29 cells. The phosphorylation of mTOR, STAT3 and GSK3β were significantly decreased when bilirubin was present. Finally, in vivo experiment exhibited the anti-tumor effect of bilirubin in the MC38 bearing mice model, but did not show tissue damage in different organs. In conclusion, bilirubin was identified as a novel P2X7R antagonist and it may have potential for anti-cancer treatment, although various functions of the molecule should be considered.

An Aerolysin-like Pore-Forming Protein Complex Targets Viral Envelope to Inactivate Herpes Simplex Virus Type 1.

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that βγ-crystallin fused aerolysin-like protein and trefoil factor complex (βγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. βγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, βγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the βγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that βγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the βγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of βγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that βγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.

Molecular Mechanism of Ultrasound-Induced Structural Defects in Liposomes: A Nonequilibrium Molecular Dynamics Simulation Study

The use of ultrasound in combination with liposomes is a promising approach to improve drug delivery. To achieve an optimal drug release rate, it is important to understand how ultrasound induces pathways on the liposome surface where drugs can be released from the liposome. To this end, we carry out large-scale ultrasound-induced molecular dynamics simulations for three single lipid component liposomes formed from the commonly used phospholipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), or phosphatidylcholine (POPC). The results show that ultrasound induces the detachment of two leaflets of the DOPC surface, suggesting that the drug release pathway may be through the low lipid packing areas on the stretched surface. In contrast, ultrasound induces pore formation on the surface of DPPC and DOPC, where drugs could escape from the liposomes. While the leaflet detachment and transient pore formation are the mechanisms of DOPC and DPPC, respectively, in both liquid-ordered and liquid-disordered phases, the leaflet detachment mechanism is switched to the transient pore formation mechanism on going from the liquid-ordered phase to the liquid-disordered phase in the POPC liposome. By adding 30% mol cholesterol, the leaflet detachment mechanism is observed in all liposomes. We found that the molecular origin that determines a mechanism is the competition between the intraleaflet and interleaflet interacting energy of lipids. The connection to experimental and theoretical modeling is discussed in some detail.

Effects of osmotic pressure on the irreversible electroporation in giant lipid vesicles.

Irreversible electroporation (IRE) is a nonthermal tumor/cell ablation technique in which a series of high-voltage short pulses are used. As a new approach, we aimed to investigate the rupture of giant unilamellar vesicles (GUVs) using the IRE technique under different osmotic pressures (Π), and estimated the membrane tension due to Π. Two categories of GUVs were used in this study. One was prepared with a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol (chol) for obtaining more biological relevance while other with a mixture of DOPG and DOPC, with specific molar ratios. We determined the rate constant (kp) of rupture of DOPG/DOPC/chol (46/39/15)-GUVs and DOPG/DOPC (40/60)-GUVs induced by constant electric tension (σc) under different Π. The σc dependent kp values were fitted with a theoretical equation, and the corresponding membrane tension (σoseq) at swelling equilibrium under Π was estimated. The estimated membrane tension agreed well with the theoretical calculation within the experimental error. Interestingly, the values of σoseq were almost same for both types of synthesized GUVs under same osmotic pressure. We also examined the sucrose leakage, due to large osmotic pressure-induced pore formation, from the inside of DOPG/DOPC/chol(46/39/15)-GUVs. The estimated membrane tension due to large Π at which sucrose leaked out was very similar to the electric tension at which GUVs were ruptured without Π. We explained the σc and Π induced pore formation in the lipid membranes of GUVs.

Comparing the interaction of the antibiotic levofloxacin with zwitterionic and anionic membranes: Calorimetry, fluorescence, and spin label studies

The present work compares the interaction of the antibiotic levofloxacin (LVX) with zwitterionic and anionic liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), respectively. By using differential scanning calorimetry (DSC), and with spin labels incorporated into liposomes at two different depths of the bilayers, we investigated the changes induced on the membrane by increasing concentrations of LVX. Further information was obtained using intrinsic LVX fluorescence. Under the conditions used here, all techniques evinced that LVX has little affinity for DPPC zwitterionic membrane. Opposite to that, LVX exhibits a considerable affinity for anionic bilayers, with membrane partition constants Kp = (3.3 ± 0.5) × 102 and (4.5 ± 0.3) × 102, for gel and fluid DPPG membranes, respectively. On binding to DPPG, LVX seems to give rise to the coexistence of LVX -rich and -poor domains on DPPG membranes, as detected by DSC. At the highest LVX concentration used (20 mol%), DSC trace shows an increase in the cooperativity of DPPG gel-fluid transition, also detected by spin labels as an increase in the bilayer packing. Moreover, LVX does not induce pore formation in either DPPG or POPG vesicles. Considering the possible relevance of LVX-membrane interaction for the biological and toxicological action of the antibiotic, the findings discussed here certainly contribute to a better understanding of its action, and the planning of new drugs.

Enhanced ammonium removal on biochar from a new forestry waste by ultrasonic activation: Characteristics, mechanisms and evaluation.

The adsorption treatment of ammonium-containing wastewater has attracted significant global attention. Most enhanced adsorption methods employ chemical modification, and there are few reports on physical activation. We present a physical activation to explore whether physical ultrasound may enhance the adsorption performance and comprehensive utilisation of a new forestry waste, Caragana korshinskii was used as a feedstock to prepare activated biochar (ACB) by controlling the pyrolysis temperatures and ultrasound parameters. The optimal parameters were determined via batch adsorption of NH4+, and the adsorption characteristics were assessed by 8 kinds of models and influence experiments. Moreover, the physicochemical properties of ACB during the pyrolysis process were investigated, and the ultrasonic activation and adsorption mechanisms were discussed using multiple characterisation techniques. Additionally, the cost analysis, the safety of the ultrasonic process and disposal method also were evaluated. The results showed that the ultrasonic activation significantly enhanced the NH4+ adsorption efficiency of biochar by approximately 5 times. ACB exhibited the best performance at 500°C with an ultrasonic activation time of 480min, frequency of 45kHz, and power of 700W. The ultrasonic activation reduced the biochar ash and induced pore formation, which increased the specific surface area through cavitation corrosion and micro-acoustic flow mechanism. The NH4+ adsorption mechanisms comprised physicochemical processes, of which physical adsorption was dominant. The preparation cost of 1kg ACB was about 0.42 US dollar, and no secondary pollution occurred in the activation process. The findings prove that ultrasonic technology is efficient and convenient for enhancing biochar adsorption performance, and thus is suitable for industrial applications and promotion.

How arginine derivatives alter the stability of lipid membranes: dissecting the roles of side chains, backbone and termini

Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R_n and of an arginine side chain mimic R_mathrm {Side} to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R_mathrm {Side} but not R_n reduces the line tension of a membrane with anionic lipids. While R_n peptides form a layer on top of a partly negatively charged lipid bilayer, R_mathrm {Side} leads to its disintegration. Complementary, our simulations show R_mathrm {Side} causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R_8 similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R_n and R_mathrm {Side} but not short R_n reduce the free energy of nucleating a pore. In short R_n, the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer mathrm {R}_{n} are membrane-active.

A broad-spectrum novel bacteriocin produced by Lactobacillus plantarum SHY 21–2 from yak yogurt: Purification, antimicrobial characteristics and antibacterial mechanism

This research aimed to find a natural biological preservative that inhibits harmful bacteria in the food production process. We screened a bacteriocin-producing Lactobacillus plantarum SHY 21–2, which was isolated from yak yogurt. The bacteriocin was purified by salting out, AKTA purifier 10 and reversed-phase high-performance liquid chromatography (RP-HPLC). The LC-MS/MS analysis revealed that the molecular weight of bacteriocin was 1362.84 Da and the predicted the amino acid sequence was RPNEPRLLLPR. This strain produced a novel IId bacteriocin and named plantaricin LP 21–2. LP 21–2 was heat-resistant and still had 96% antibacterial activity after being exposed to 121 °C for 15 min. LP 21-2 had obvious antibacterial activity at pH 2–5 and could be completely inactivated by pepsin, trypsin, and papain. LP 21-2 had a broad antimicrobial spectrum against on Gram-positive bacteria, Gram-negative bacteria and fungi, including Staphylococcus aureus ATCC25923, Salmonella typhi CMCC50071 and Saccharomyces cerevisiae ATCC9763. The minimal inhibitory concentration (MIC) of LP 21–2 on Staphylococcus aureus ATCC 25923 was 0.18 mg/ml. Analyzed by scanning electron microscopy and transmission electron microscopy, LP 21–2 induced pore formation in cell membranes and caused cytoplasmic leakage to kill bacteria. These results indicate that plantaricin LP 21-2 has potential as a bioprotective agent in food.

Fabrication and Characterization of Novel Poly(D-lactic acid) Nanocomposite Membrane for Water Filtration Purpose.

The development of membrane technology from biopolymer for water filtration has received a great deal of attention from researchers and scientists, owing to the growing awareness of environmental protection. The present investigation is aimed at producing poly(D-lactic acid) (PDLA) membranes, incorporated with nanocrystalline cellulose (NCC) and cellulose nanowhisker (CNW) at different loadings of 1 wt.% (PDNC-I, PDNW-I) and 2 wt.% (PDNC-II PDNW-II). From morphological characterization, it was evident that the nanocellulose particles induced pore formation within structure of the membrane. Furthermore, the greater surface reactivity of CNW particles facilitates in enhancing the surface wettability of membranes due to increased hydrophilicity. In addition, both thermal and mechanical properties for all nanocellulose filled membranes under investigation demonstrated significant improvement, particularly for PDNW-I-based membranes, which showed improvement in both aspects. The membrane of PDNW-I presented water permeability of 41.92 L/m2h, when applied under a pressure range of 0.1–0.5 MPa. The investigation clearly demonstrates that CNWs-filled PDLA membranes fabricated for this investigation have a very high potential to be utilized for water filtration purpose in the future.

High-speed atomic force microscopy to study pore-forming proteins.

Pore-forming proteins (PFPs) include virulence factors that are produced by many pathogenic bacteria. However, PFPs also comprise non-virulence factors, such as apoptotic Bcl2-like proteins, and also occur in non-pathogenic bacteria and indeed in all kingdoms of life. Pore-forming proteins are an ancient class of proteins, which are tremendously powerful in damaging cell membranes. In general, upon binding to lipid membranes, they convert from the soluble monomeric form into an oligomeric state, and then undergo a dramatic conformational change to form transmembrane pores. Thus, PFPs render the plasma membrane of their target cells permeable to solutes, potentially leading to cell death, or to more subtle manipulations of cellular functions. Recent cryo-EM and X-ray crystallography studies revealed high-resolution structures of several PFPs in their pre-pore and pore states, however many aspects regarding the cues that induce pore formation, the pre-pore to pore conformational transition, the mechanism of membrane permeation and associated dynamics are still less well understood, and direct visualization of the dynamics of these transitions are missing. Using high-speed atomic force microscopy (HS-AFM), the kinetics of oligomerization and the pre-pore to pore transition dynamics of various PFPs, such as Listeriolysin O (LLO), lysenin, and Perforin-2 (PFN2), could be studied. These studies revealed that LLO does not form pores of regular shape or size, but rather forms membrane inserted arcs that propagate and damage lipid membranes as lineactants. In contrast, lysenin forms stable pre-pore and pore nonameric rings and HS-AFM allowed to study their diffusion on and the pH-dependent insertion into the membrane. Similarly, PFN2 underwent pre-pore to pore transition upon acidification. The openness of the HS-AFM system allowed the transition to be imaged in real time and revealed that all observed molecules transitioned into the pore state within 3s. In this chapter, we detail protocols to prepare lipids, form supported lipid bilayers, and provide guidelines for real-time, real-space HS-AFM observations of PFPs in action.

Humanin selectively prevents the activation of pro-apoptotic protein BID by sequestering it into fibers

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into β-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to β-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.

Interaction of Ordered Lipid Domain Boundaries and Amphipathic Peptides Regulates Probability of Pore Formation in Membranes

Biological membranes include various lipids. A heterogeneity of lipid composition can lead to phase separation resulting in the formation of ordered lipid domains, which differ in lipid composition from the other (disordered) part of the membrane. Membrane deformations, which occur at the domain boundary, can affect the lateral distribution of various membrane inclusions. In this paper, in the framework of theory of elasticity of lipid membranes, the influence of the boundaries of lipid domains on the lateral distribution of amphipathic peptides adsorbed on the membrane is considered. Such peptides can cause the formation of through pores. We showed that with an increase in the concentration of amphipathic peptides on the membrane, they first line up near the boundary of the domain parallel to it, thus losing the ability to induce pore formation. Then, as the domain boundary is completely occupied, new peptides stand parallel to the line of peptides that are already at the boundary, at a distance of about 5 nm from the boundary. In this configuration, the probability of formation of through pores in the membrane is increased. Besides, it is shown that the spontaneous curvature of monolayers of the ordered domain and disordered membrane determines the energy of incorporation of peptides into the membrane and their distribution between the phases. However, the spontaneous curvature scarcely influences the interaction of amphipathic peptides with the boundary of the ordered domain.