scholarly journals Panorama of the Intracellular Molecular Concert Orchestrated by Actinoporins, Pore-Forming Toxins from Sea Anemones

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 567
Author(s):  
Carlos Alvarez ◽  
Carmen Soto ◽  
Sheila Cabezas ◽  
Javier Alvarado-Mesén ◽  
Rady Laborde ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Conformational Changes ◽  
Pore Formation ◽  
Eukaryotic Cells ◽  
Sea Anemones ◽  
Vaccine Platform ◽  
The Caribbean ◽  
Pore Forming Proteins ◽  
Novel Applications ◽  
Intracellular Action

Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs’ intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.

CAN SINGLE ELECTRONS INITIATE FUSION OF BIOLOGICAL MEMBRANES?

2007 ◽  
Vol 02 (01) ◽  
pp. 23-31 ◽  
Author(s):  
MARTIN KOCH ◽  
ANGELA M. OTTO ◽  
JOACHIM WIEST ◽  
BERNHARD WOLF
Keyword(s):  
Conformational Changes ◽  
Pore Formation ◽  
Aromatic Amino Acids ◽  
Fusion Pore ◽  
Mathematical Approach ◽  
Animal Cells ◽  
Lipid Complex ◽  
Single Electrons ◽  
Initial Event ◽  
Membrane Barrier

Animal cells export the content of vesicles by exocytosis, a process in which a vesicle and the plasma membrane fuse and ultimately form a pore. The initial event leading to the breakthrough of the membranes for pore formation is not well understood. Here we present a mathematical approach which suggests that this process is initiated by single electrons which could be present in the immediate proximity of the fusing membranes in aromatic amino acids. The electron can be regarded as non-classical; it takes up energy and transfers it to the membrane barrier, thereby eliciting conformational changes in the proteo-lipid complex leading to membrane fusion and thus initiating the opening of the fusion pore.

Genetically-Engineered Protease-Activated Triggers in a Pore-Forming Protein

1993 ◽  
Vol 330 ◽  
Author(s):  
Barbara Walker ◽  
Nathan Walsh ◽  
Hagan Bayley
Keyword(s):  
Genetic Engineering ◽  
Cancer Cells ◽  
Pore Formation ◽  
Polypeptide Chain ◽  
Genetically Engineered ◽  
Recognition Sequence ◽  
Selective Activation ◽  
Selective Destruction ◽  
Pore Forming Proteins ◽  
Central Loop

ABSTRACTProtease-activated triggers have been introduced Into a pore-forming protein, staphylococcal a-hemolysin (αHL). The hemolysin was remodeled by genetic engineering to form two-chain constructs with redundant polypeptide sequences at the central loop, the Integrity of which Is crucial for efficient pore formation. The new hemolysins are activated when the polypeptide extensions are removed by proteases. By alterating the protease recognition sequence in the loop, selective activation by specified proteases can be obtained. Protease-triggered pore-forming proteins might be used for the selective destruction of cancer cells that bear tumor-associated proteases. When certain two-chain constructs are treated with proteases, a full-length polypeptide chain forms as the result of a protease-mediated transpeptidation reaction. This reaction might be used to produce chimeric hemolysins that are Inaccessible by conventional routes.

scholarly journals Proteasomal conformation controls unfolding ability

2021 ◽  
Vol 118 (25) ◽  
pp. e2101004118
Author(s):  
Julianna R. Cresti ◽  
Abramo J. Manfredonia ◽  
Christopher E. Bragança ◽  
Joseph A. Boscia ◽  
Christina M. Hurley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational State ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Ubiquitinated Protein ◽  
Equilibrium Conditions ◽  
Substrate Processing

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome’s various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome’s ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome’s conformational state and its unfolding ability.

scholarly journals Pore formation by actinoporins, cytolysins from sea anemones

2016 ◽  
Vol 1858 (3) ◽  
pp. 446-456 ◽  
Author(s):  
Nejc Rojko ◽  
Mauro Dalla Serra ◽  
Peter Maček ◽  
Gregor Anderluh
Keyword(s):  
Pore Formation ◽  
Sea Anemones

scholarly journals Membrane insertion of the N-terminal α-helix of equinatoxin II, a sea anemone cytolytic toxin

2004 ◽  
Vol 384 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Ion GUTIÉRREZ-AGUIRRE ◽  
Ariana BARLIČ ◽  
Zdravko PODLESEK ◽  
Peter MAČEK ◽  
Gregor ANDERLUH ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Lipid Membrane ◽  
Model Systems ◽  
Tryptophan Residue ◽  
Sea Anemones ◽  
Fluorescence Measurements ◽  
Tryptophan Residues ◽  
Equinatoxin Ii ◽  
Α Helix ◽  
Pore Forming Proteins

Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic α-helix. In the present study, we have investigated the transfer of the N-terminal α-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic α-helix (Ile18→Trp, Val22→Trp and Ala25→Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22→Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.

scholarly journals Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins

2019 ◽  
Vol 116 (26) ◽  
pp. 12839-12844 ◽  
Author(s):  
Ilanila I. Ponmalar ◽  
Ramesh Cheerla ◽  
K. Ganapathy Ayappa ◽  
Jaydeep K. Basu
Keyword(s):  
Conformational Changes ◽  
Pore Formation ◽  
Bacterial Infections ◽  
Cell Lysis ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Listeriolysin O ◽  
Conformational States ◽  
Wide Range ◽  
Membrane Bound

Pore-forming toxins (PFTs) are a class of proteins implicated in a wide range of virulent bacterial infections and diseases. These toxins bind to target membranes and subsequently oligomerize to form functional pores that eventually lead to cell lysis. While the protein undergoes large conformational changes on the bilayer, the connection between intermediate oligomeric states and lipid reorganization during pore formation is largely unexplored. Cholesterol-dependent cytolysins (CDCs) are a subclass of PFTs widely implicated in food poisoning and other related infections. Using a prototypical CDC, listeriolysin O (LLO), we provide a microscopic connection between pore formation, lipid dynamics, and leakage kinetics by using a combination of Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) measurements on single giant unilamellar vesicles (GUVs). Upon exposure to LLO, two distinct populations of GUVs with widely different leakage kinetics emerge. We attribute these differences to the existence of oligomeric intermediates, sampling various membrane-bound conformational states of the protein, and their intimate coupling to lipid rearrangement and dynamics. Molecular dynamics simulations capture the influence of various membrane-bound conformational states on the lipid and cholesterol dynamics, providing molecular interpretations to the FRET and FCS experiments. Our study establishes a microscopic connection between membrane binding and conformational changes and their influence on lipid reorganization during PFT-mediated cell lysis. Additionally, our study provides insights into membrane-mediated protein interactions widely implicated in cell signaling, fusion, folding, and other biomolecular processes.

scholarly journals Haemolytic actinoporins interact with carbohydrates using their lipid-binding module

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160216 ◽  
Author(s):  
Koji Tanaka ◽  
Jose M. M. Caaveiro ◽  
Koldo Morante ◽  
Kouhei Tsumoto
Keyword(s):  
Protein Interactions ◽  
Pore Formation ◽  
Plasma Membranes ◽  
Lipid Binding ◽  
Target Cells ◽  
Sea Anemones ◽  
Molecular Systems ◽  
Membrane Pores ◽  
Living Organisms ◽  
Lipid Protein Interactions

Pore-forming toxins (PFTs) are proteins endowed with metamorphic properties that enable them to stably fold in water solutions as well as in cellular membranes. PFTs produce lytic pores on the plasma membranes of target cells conducive to lesions, playing key roles in the defensive and offensive molecular systems of living organisms. Actinoporins are a family of potent haemolytic toxins produced by sea anemones vigorously studied as a paradigm of α-helical PFTs, in the context of lipid–protein interactions, and in connection with nanopore technologies. We have recently reported that fragaceatoxin C (FraC), an actinoporin, engages biological membranes with a large adhesive motif allowing the simultaneous attachment of up to four lipid molecules prior to pore formation. Since actinoporins also interact with carbohydrates, we sought to understand the molecular and energetic basis of glycan recognition by FraC. By employing structural and biophysical methodologies, we show that FraC engages glycans with low affinity using its lipid-binding module. Contrary to other PFTs requiring separate domains for glycan and lipid recognition, the small single-domain actinoporins economize resources by achieving dual recognition with a single binding module. This mechanism could enhance the recruitment of actinoporins to the surface of target tissues in their marine environment. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

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

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160212 ◽  
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’.

scholarly journals The DNA damage-recognition problem in human and other eukaryotic cells: the XPA damage binding protein

1997 ◽  
Vol 328 (1) ◽  
pp. 1-12 ◽  
Author(s):  
E. James CLEAVER ◽  
J. Christopher STATES
Keyword(s):  
Zinc Finger ◽  
Conformational Changes ◽  
Binding Capacity ◽  
Binding Protein ◽  
Protein A ◽  
Eukaryotic Cells ◽  
Damage Recognition ◽  
Recognition Ability ◽  
Associated Proteins ◽  
Group A

The capacity of human and other eukaryotic cells to recognize a disparate variety of damaged sites in DNA, and selectively excise and repair them, resides in a deceptively small simple protein, a 38-42 kDa zinc-finger binding protein, XPA (xeroderma pigmentosum group A), that has no inherent catalytic properties. One key to its damage-recognition ability resides in a DNA-binding domain which combines a zinc finger and a single-strand binding region which may infiltrate small single-stranded regions caused by helix-destabilizing lesions. Another is the augmentation of its binding capacity by interactions with other single-stranded binding proteins and helicases which co-operate in the binding and are unloaded at the binding site to facilitate further unwinding of the DNA and subsequent catalysis. The properties of these reactions suggest there must be considerable conformational changes in XPA and associated proteins to provide a flexible fit to a wide variety of damaged structures in the DNA.

scholarly journals Coordinated conformational changes in the V1 complex during V-ATPase reversible dissociation

2021 ◽  
Author(s):  
Thamiya Vasanthakumar ◽  
Kristine A Keon ◽  
Stephanie A Bueler ◽  
Michael C Jaskolka ◽  
John L Rubinstein
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Rotational State ◽  
Eukaryotic Cells ◽  
Subunit C ◽  
Unknown Mechanism ◽  
Conformational Rearrangement ◽  
Reversible Dissociation ◽  
Intracellular Compartments

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited upon disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the RAVE complex through an unknown mechanism. We used cryoEM of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex, and the V1 complex lacking subunit C. Upon separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.

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