Incomplete pneumolysin oligomers form membrane pores

Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.

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The mechanisms of action of water-soluble aminohexanoic and malonic adducts of fullerene C60 with hexamethonium on model lipid membranes

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2020.183433 ◽

2020 ◽

Vol 1862 (11) ◽

pp. 183433

Author(s):

S.S. Efimova ◽

D.A. Khaleneva ◽

E.V. Litasova ◽

L.B. Piotrovskiy ◽

O.S. Ostroumova

Keyword(s):

Lipid Membranes ◽

Mechanisms Of Action ◽

Fullerene C60 ◽

Water Soluble ◽

Model Lipid Membranes

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Electrodes modified with lipid membranes to study quinone oxidoreductases

Biochemical Society Transactions ◽

10.1042/bst0370707 ◽

2009 ◽

Vol 37 (4) ◽

pp. 707-712 ◽

Cited By ~ 7

Author(s):

Sophie A. Weiss ◽

Lars J.C. Jeuken

Keyword(s):

Lipid Membranes ◽

Biochemical Properties ◽

Model Membranes ◽

Water Soluble ◽

Enzyme Assays ◽

Membrane Enzymes ◽

Membrane Bound ◽

Hydrophobic Nature

Quinone oxidoreductases are a class of membrane enzymes that catalyse the oxidation or reduction of membrane-bound quinols/quinones. The conversion of quinone/quinol by these enzymes is difficult to study because of the hydrophobic nature of the enzymes and their substrates. We describe some biochemical properties of quinones and quinone oxidoreductases and then look in more detail at two model membranes that can be used to study quinone oxidoreductases in a native-like membrane environment with their native lipophilic quinone substrates. The results obtained with these model membranes are compared with classical enzyme assays that use water-soluble quinone analogues.

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Repurposing a pore: highly conserved perforin-like proteins with alternative mechanisms

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0212 ◽

2017 ◽

Vol 372 (1726) ◽

pp. 20160212 ◽

Cited By ~ 11

Author(s):

Tao Ni ◽

Robert J. C. Gilbert

Keyword(s):

Retinoic Acid ◽

Bone Morphogenetic Protein ◽

Pore Formation ◽

Membrane Attack Complex ◽

Common Mechanism ◽

Developmental Processes ◽

Membrane Pores ◽

Morphogenetic Protein ◽

Specific Proteins ◽

Pore Forming Proteins

Pore-forming proteins play critical roles in pathogenic attack and immunological defence. The membrane attack complex/perforin (MACPF) group of homologues represents, with cholesterol-dependent cytolysins, the largest family of such proteins. In this review, we begin by describing briefly the structure of MACPF proteins, outlining their common mechanism of pore formation. We subsequently discuss some examples of MACPF proteins likely implicated in pore formation or other membrane-remodelling processes. Finally, we focus on astrotactin and bone morphogenetic protein and retinoic acid-induced neural-specific proteins, highly conserved MACPF family members involved in developmental processes, which have not been well studied to date or observed to form a pore—and which data suggest may act by alternative mechanisms. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

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Molecular Mobility in Trehalose Loaded Mammalian Cells: Time-Resolved Fluorescence Anisotropy Measurements

ASME 2008 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2008-193077 ◽

2008 ◽

Author(s):

Nilay Chakraborty ◽

Wesley Parker ◽

Kevin E. Elliott ◽

Stuart T. Smith ◽

Patrick J. Moyer ◽

...

Keyword(s):

Mammalian Cells ◽

Fluid Phase ◽

Water Soluble ◽

Great Promise ◽

Time Resolved ◽

Intracellular Space ◽

Membrane Pores ◽

Fluid Phase Endocytosis ◽

Membrane Bound ◽

Cellular Compartments

Many preservation methods have utilized sugars such as trehalose as protectants against injury during cell preservation processing, especially during drying (1–5). As mammalian cells do not synthesize trehalose, research in the mammalian cell desiccation field has focused on the development of strategies to enable trehalose delivery into the intracellular milieu. Numerous techniques have been explored ranging from microinjection (2) to the creation or utilization of membrane pores (1,3). Fluid phase endocytosis has shown great promise as an effective strategy for non-invasively delivering water-soluble materials into the intracellular space (4, 5). In this technique trehalose is transported across the cell membrane in membrane-bound cellular compartments called endosomes. Cells incubated in cell culture medium containing trehalose have been shown to take up considerable amounts of trehalose by this technique (4, 5). How much of this trehalose actually become available for protection of biomolecules during the dehydration process has yet to be determined.

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An effect of antibiotic amphotericin B on ion transport across model lipid membranes and tonoplast membranes

Biochemical Pharmacology ◽

10.1016/j.bcp.2005.06.001 ◽

2005 ◽

Vol 70 (5) ◽

pp. 668-675 ◽

Cited By ~ 24

Author(s):

Monika Hereć ◽

Halina Dziubińska ◽

Kazimierz Trębacz ◽

Jacek W. Morzycki ◽

Wiesław I. Gruszecki

Keyword(s):

Ion Transport ◽

Amphotericin B ◽

Lipid Membranes ◽

Model Lipid Membranes

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Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement

Biochemical Journal ◽

10.1042/bj2840169 ◽

1992 ◽

Vol 284 (1) ◽

pp. 169-176 ◽

Cited By ~ 60

Author(s):

T R Hughes ◽

S J Piddlesden ◽

J D Williams ◽

R A Harrison ◽

B P Morgan

Keyword(s):

Affinity Purification ◽

Membrane Attack Complex ◽

Erythrocyte Membranes ◽

Dependent Manner ◽

Inhibitory Protein ◽

Rat Erythrocytes ◽

Butanol Extract ◽

Isolation And Characterization ◽

Membrane Bound

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated ‘reactive’ lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

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