Alzheimer's Disease (AD) is a devastating neurodegenerative disease that is characterized by brain atrophy, neuronal and synaptic loss, cognitive decline, trouble handling activities of daily life, and ultimately leads to death. Worldwide, at least 30 million people suffer from AD, with 5.8 million suffering in the US alone. Despite extensive basic and clinical research, the underlying molecular mechanisms behind AD remain largely unknown. There are four FDA-approved compounds are used for alleviating symptoms but have no curative potency. The first potentially disease-modifying AD drug, aducanumb, was approved by FDA in June 2021. The main histopathological traits of AD are the Amyloid-beta (Aβ) peptide and the tau protein. Aβ aggregates to form extracellular plaques in brain parenchyma and vasculature while tau forms intraneuronal tangles. Aβ is produced by enzymatic cleavage of the amyloid precursor protein (APP) in the brain. Once APP cleavage occurs, Ab monomers either aggregate extracellularly to form buildups of sticky plaque or embed themselves within the neuronal cell membrane to form pores, causing homeostatic dysregulation and eventually cell death. The mechanism of membrane pores formed by Ab and the pore structure remain to be characterized. This study aims to analyze the structure of four Aβ species in lipid membranes. These are the most abundant form of Aβ, Aβ1-40, and the more cytotoxic form, Aβ1-42, as well as their pyroglutamylated counterparts, pEAβ3-40 and pEAβ3-42, which are hypertoxic. These peptides have been studied using biophysical approaches, i.e., circular dichroism, fluorescence spectroscopy, and Fourier transform infrared spectroscopy. Elucidation of the structure of Aβ membrane pores provides valuable insight into the mechanism of Aβ toxicity and may help develop novel therapies for the lethal mystery that is AD.

No description available.

No / Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel phase and in the DMSO-induced fluidized state. Our simulations show that the fluid phase bilayers form archetypal water-filled hydrophilic pores similar to those observed in phospholipid bilayers. In contrast, the rigid gel-phase bilayers develop hydrophobic pores. At the relatively small pore diameters studied here, the hydrophobic pores are empty rather than filled with bulk water, suggesting that they do not compromise the barrier function of ceramide membranes. A phenomenological analysis suggests that these vapor pores are stable, below a critical radius, because the penalty of creating water-vapor and tail-vapor interfaces is lower than that of directly exposing the strongly hydrophobic tails to water. The PMCF free energy profile of the vapor pore supports this analysis. The simulations indicate that high DMSO concentrations drastically impair the barrier function of the skin by strongly reducing the free energy required for pore opening. / EPSRC

Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophagelocalized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1 (pro-IL-1)-degrading caspase-1 to lead to IL-1 release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.

<p> Bax is a pro-apoptotic protein believed to permeabilize mitochondria during apoptosis. The mechanism Bax uses is not well understood. In this work, we use fluorescence techniques to shed light on how tBid activates Bax and we examine the topology of the pore-forming domain of Bax. </p> <p> The manner in which tBid promotes apoptosis via Bax activation is not known. Study of tBid and Bax interaction using a new FRET pair showed that the proteins only interacted in the presence ofmembranes. The Bax pore was shown to have a variable size distribution. A fluorescence technique of simultaneously measuring pore formation, Bax insertion and FRET showed that tBid interaction with Bax occurred before all the Bax inserted or formed pores in the liposomes. A chronological order is proposed for Bax pore formation. tBid first binds to liposomes. tBid proceeds to interact with Bax, and Bax inserts into the membrane. After insertion, Bax oligomerizes and forms small pores. More Bax is recruited and the pores become larger. </p> <p> The two central hairpin helices of Bax, helices 5 and 6, are known as the pore-forming domain. We used cysteine scanning with the environment sensitive fluoroprobe NBD to gain insight into the topology of these helices. Fluorescence intensity changes and emission blue shifts showed that residues in these helices undergo conformational reorganization during pore formation. In the activated oligomeric conformation, fluorescence lifetimes showed that helix 5 was more inaccessible to water than helix 6. Cobalt, a cationic NBD quencher, effectively quenched residues in the pore-forming domain, consistent with a pore that is lined with anionic lipid head groups. Quenching with nitroxide groups at various lipid depths showed that residues on helix 6 were most quenched by a shallow quencher, while residues on helix 5 were quenched by deeper quenchers. Compared to beta sheet pore-forming proteins, the data obtained suggests that Bax and possibly other alpha helical pore-forming proteins form a lipidic pore in a dynamic environment. Combined together, the data suggest a model for Bax in which helix 5 spans the bilayer, and helix 6 is buried just below the lipid headgroups of a toroidal pore. </p> / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23517号 / 工博第4929号 / 新制||工||1769(附属図書館) / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 佐野 紀彰, 教授 宮原 稔, 教授 河瀬 元明 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

no / Review article. No abstract.

Recent experimental studies indicate that polymeric structures with a well-adjusted balance of amphiphilic parts may translocate through self-assembled phospholipid bilayers and enhance the passive trans-membrane transport of smaller molecules. Using a coarse grained lattice Monte Carlo model with explicit solvent we investigate self-assembled lipid bilayers interacting with a linear polymer chain under variation of the hydrophobicity of the chain. Here, we focus on the relationship between the chain's hydrophobicity and its translocation behavior through the membrane as well as induced membrane perturbations. We show, that there is an adsorption transition of the polymer at the bilayer interface, where effectively the solvent phase and the tail phase of the bilayer are equally repulsive for the polymer. Close to this adsorption threshold of the polymer both the translocation probability of the polymer as well as the permeability of the membrane with respect to solvent are enhanced significantly. The frequency of polymer translocation events can be understood quantitatively assuming a simple diffusion along a one-dimensional free energy profile, which is controlled by the effective lipophilicity of the chain and the tail-packing in the bilayer's core. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

Les toxines Cry sont des protéines synthétisées sous forme de cristaux par la bactérie bacille de Thuringe pendant la sporulation. Elles sont largement utilisées comme agents de lutte biologique, car elles sont toxiques envers plusieurs espèces d’invertébrées, y compris les nématodes. Les toxines Cry5B sont actives contre certaines espèces de nématodes parasites, y compris Ankylostoma ceylanicum un parasite qui infeste le système gastro-intestinal des humains. Jusqu’au présent, le mode d’action des toxines Cry nématicides reste grandement inconnu, sauf que leurs récepteurs spécifiques sont des glycolipides et qu’elles causent des dommages importants aux cellules intestinales. Dans cette étude, on démontre pour la première fois que la toxine nématicide Cry5Ba, membre de la famille des toxines à trois domaines et produite par la bactérie bacille de Thuringe, forme des pores dans les bicouches lipidiques planes en absence de récepteurs. Les pores formés par cette toxine sont de sélectivité cationique, à pH acide ou alcalin. Les conductances des pores formés sous conditions symétriques de 150 mM de KCl varient entre 17 et 330 pS, à pH 6.0 et 9.0. Les niveaux des conductances les plus fréquemment observés diffèrent les uns des autres par environ 17 à 18 pS, ce qui est compatible avec l’existence d’arrangement d’un nombre différent de pores élémentaires similaires, activés de façon synchronisée, ou avec la présence d’oligomères de tailles variables et de différents diamètres de pores. / Cry toxins are proteins synthetized as crystal inclusions by the Bacillus thuringiensis bacterium upon sporulation. They are used widely as biological control agents, as they exhibit toxicity to a range of invertebrates, including nematodes. The Cry5B toxins are active against a number of parasitic nematode species, such as Ancylostoma ceylanicum a human gastro-intestinal parasite. So far, the mode of action of nematicidal Cry toxins is largely unknown, except for the facts that their specific receptors are glycolipids and that they cause prominent damage to nematode intestinal cells. In this study, we show for the first time that the nematicidal Cry5Ba toxin, a member of the three domain family of toxins produced by the Bacillus thuringiensis forms pores in receptor-free planar lipid bilayers. The pores formed by the toxin were cation selective, both under acid and alkaline pH conditions. Under symmetrical 150 mM KCl conditions, pore activity was characterized by conductances ranging from 17 to 330 pS, at both pH 6.0 and 9.0. The most frequently observed conductance levels differed from each other by approximately 17 to 18 pS consistent with the existence of clusters of different number of elementary, similar, co-operatively gated pores, or with the presence of variable size oligomers with different pore diameters.