Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of β-barrel pore-forming toxins (β-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane β-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO.

In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens.

Littoporins: Novel actinoporin-like proteins in caenogastropod genus Littorina.

In the course of searching for genes controlling the immune system in caenogastropod mollusks, we characterized and phylogenetically placed five new actinoporin-like cytolysins expressed in periwinkles of the genus Littorina. These newly discovered proteins, named littoporins (LitP), contain a central cytolysin/lectin domain and exhibit a predicted protein fold that is almost identical to the three-dimensional structures of actinoporins. Two of these proteins, LitP-1 and LitP-2, were found to be upregulated in L.

Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization.

Programmable de novo designed coiled coil-mediated phase separation in mammalian cells.

Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. The design of liquid-liquid phase separated (LLPS) condensates based on de novo designed tunable modules that interact in a well-understood, controllable manner could improve our understanding of this phenomenon and enable the introduction of new features. Here we report the construction of CC-LLPS in mammalian cells, based on designed coiled-coil (CC) dimer-forming modules, where the stability of CC pairs, their number, linkers, and sequential arrangement govern the transition between diffuse, liquid and immobile condensates and are corroborated by coarse-grained molecular simulations.

A label-free approach for measuring interactions of proteins with lipid membranes.

In this issue of Cell Reports Methods, Sadi et al. present a nuclear magnetic resonance approach for quantitative assessment of protein interactions with lipid membranes. It is sensitive, applicable to diverse membrane systems, covers a broad range of Ks, and does not require large amounts of material.

Structural basis for the unique molecular properties of broad-range phospholipase C from Listeria monocytogenes.

Listeriosis is one of the most serious foodborne diseases caused by the intracellular bacterium Listeria monocytogenes. Its two major virulence factors, broad-range phospholipase C (LmPC-PLC) and the pore-forming toxin listeriolysin O (LLO), enable the bacterium to spread in the host by destroying cell membranes. Here, we determine the crystal structure of LmPC-PLC and complement it with the functional analysis of this enzyme.

Surface plasmon resonance and microscale thermophoresis approaches for determining the affinity of perforin for calcium ions.

Perforin is a pore-forming protein that plays a crucial role in the immune system by clearing virus-infected or tumor cells. It is released from cytotoxic granules of immune cells and forms pores in targeted lipid membranes to deliver apoptosis-inducing granzymes. It is a very cytotoxic protein and is therefore adapted not to act in producing cells.

Bacillus thuringiensis toxin Cyt2Aa forms filamentous oligomers when exposed to lipid membranes or detergents.

The bacterium Bacillus thuringiensis (Bt) produces insecticidal proteins during the sporulation phase. These proteins are located in parasporal crystals consisting of two delta-endotoxin classes, crystal (Cry) and cytolytic (Cyt) toxins. In vitro, Cyt toxins show cytolytic activity against bacterial and a variety of insect and mammalian cells.

Disruption of plant plasma membrane by Nep1-like proteins in pathogen-plant interactions.

Lipid membrane destruction by microbial pore-forming toxins (PFTs) is a ubiquitous mechanism of damage to animal cells, but is less prominent in plants. Nep1-like proteins (NLPs) secreted by phytopathogens that cause devastating crop diseases, such as potato late blight, represent the only family of microbial PFTs that effectively damage plant cells by disrupting the integrity of the plant plasma membrane. Recent research has elucidated the molecular mechanism of NLP-mediated membrane damage, which is unique among microbial PFTs and highly adapted to the plant membrane environment.

Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians.

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action.

Interactions of (-)-epigallocatechin-3-gallate with model lipid membranes.

(-)-Epigallocatechin-3-gallate (EGCG) is a flavonoid known for its good antioxidant potential and health benefits. It is one of the most intriguing flavonoids, especially because of its specific interactions with model lipid membranes. It was noticed that EGCG might form EGCG rich domains/rafts at certain compositions of lipid membranes.

Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor.

DNA transcription is regulated by a range of diverse mechanisms and primarily by transcription factors that recruit the RNA polymerase complex to the promoter region on the DNA. Protein binding to DNA at nearby or distant sites can synergistically affect this process in a variety of ways, but mainly through direct interactions between DNA-binding proteins. Here we show that a Transcription Activator-Like Effector (TALE), which lacks an activation domain, can enhance transcription in mammalian cells when it binds in the vicinity of and without direct interaction with several different dimeric or monomeric transcription factors.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane.

Vesicle cholesterol controls exocytotic fusion pore.

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma.

Lipid-Binding Aegerolysin from Biocontrol Fungus .

Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis.

Beyond pore formation: reorganization of the plasma membrane induced by pore-forming proteins.

Pore-forming proteins (PFPs) are a heterogeneous group of proteins that are expressed and secreted by a wide range of organisms. PFPs are produced as soluble monomers that bind to a receptor molecule in the host cell membrane. They then assemble into oligomers that are incorporated into the lipid membrane to form transmembrane pores.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies.

Nep1-like proteins as a target for plant pathogen control.

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors.

Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells.

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes.