Ferroptosis in health and disease.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis

Pore-forming proteins: From defense factors to endogenous executors of cell death

Pore-forming proteins (PFPs) and small antimicrobial peptides (AMPs) represent a large family of molecules with the common ability to punch holes in cell membranes to alter their permeability. They play a fundamental role as infectious bacteria's defensive tools against host's immune system and as executors of endogenous machineries of regulated cell death in eukaryotic cells. Despite being highly divergent in primary sequence and 3D structure, specific folds of pore-forming domains have been conserved. In fact, pore formation is considered an ancient mechanism that takes place through a general multistep process involving: membrane partitioning and insertion, oligomerization and pore formation. However, different PFPs and AMPs assemble and form pores following different mechanisms that could end up either in the formation of protein-lined or protein-lipid pores. In this review, we analyze the current findings in the mechanism of action of different PFPs and AMPs that support a wide role of membrane pore formation in nature. We also provide the newest insights into the development of state-of-art techniques that have facilitated the characterization of membrane pores. To understand the physiological role of these peptides/proteins or develop clinical applications, it is essential to uncover the molecular mechanism of how they perforate membranes

Techniques for studying membrane pores

Pore-forming proteins (PFPs) are of special interest because of the association of their activity with the disruption of the membrane impermeability barrier and cell death. They generally convert from a monomeric, soluble form into trans-membrane oligomers that induce the opening of membrane pores. The study of pore formation in membranes with molecular detail remains a challenging endeavor because of its highly dynamic and complex nature, usually involving diverse oligomeric structures with different functionalities. Here we discuss current methods applied for the structural and functional characterization of PFPs at the individual vesicle and cell level. We highlight how the development of high-resolution and single-molecule imaging techniques allows the analysis of the structural organization of protein oligomers and pore entities in lipid membranes

Partners in Crime: The Interplay of Proteins and Membranes in Regulated Necrosis

Pyroptosis, necroptosis, and ferroptosis are well-characterized forms of regulated necrosis that have been associated with human diseases. During regulated necrosis, plasma membrane damage facilitates the movement of ions and molecules across the bilayer, which finally leads to cell lysis and release of intracellular content. Therefore, these types of cell death have an inflammatory phenotype. Each type of regulated necrosis is mediated by a defined machinery comprising protein and lipid molecules. Here, we discuss how the interaction and reshaping of these cellular components are essential and distinctive processes during pyroptosis, necroptosis, and ferroptosis. We point out that although the plasma membrane is the common target in regulated necrosis, different mechanisms of permeabilization have emerged depending on the cell death form. Pore formation by gasdermins (GSDMs) is a hallmark of pyroptosis, while mixed lineage kinase domain-like (MLKL) protein facilitates membrane permeabilization in necroptosis, and phospholipid peroxidation leads to membrane damage in ferroptosis. This diverse repertoire of mechanisms leading to membrane permeabilization contributes to define the specific inflammatory and immunological outcome of each type of regulated necrosis. Current efforts are focused on new therapies that target critical protein and lipid molecules on these pathways to fight human pathologies associated with inflammation

Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.Fil: Pedrera, Lohans. Universidad de la Habana; CubaFil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Ros, Uris. Universidad de la Habana; CubaFil: Lanio, Maria E.. Universidad de la Habana; CubaFil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Alvarez, Carlos. Universidad de la Habana; Cub

Isolation and partial purification of a hemolytic sphingomyelin-inhibitable fraction from the sea anemone Anthopleura nigrescens

Actinoporins are highly hemolytic pore-forming proteins with a molecular mass of around 20 kDa and high affinity for sphingomyelin-containing membranes. On the crude extract of the sea anemone Anthopleura nigrescens, the hemolytic activity (HA) was detected. In order to identify the presence of pore-forming proteins similar to actinoporins in this anemone, the fractionation and analysis of its crude extract was carried out. The aqueous extract of the whole body was subjected to gel filtration chromatography on Sephadex G50 medium rendering three resolved peaks (P-I, P-II, and P-III) as measured by their absorbance at 280 nm. Functional characterization of the crude extract and chromatographic fractions was evaluated by HA against human red blood cells. The crude extract and two peaks (P-II and P-III) showed HA. Interestingly, the HA of the crude extract and P-II were specifically inhibited by small unilamellar vesicles of phosphatidylcholine: sphingomyelin (1:1). Both the crude extract and P-II revealed the existence of at least one protein band around 20 kDa by SDS-PAGE. The inhibition by sphingomyelin of the whole body extract HA and the localization of this property in P-II that could be associated with a protein of around 20 kDa suggest the presence of at least one novel actinoporin in A. nigrescens. Furthermore, this is the first report of a biochemical activity for this sea anemone. Work is in progress in order to purify and characterize the molecular entities responsible for this HA inhibited by sphingomyelin.Las actinoporinas son proteínas formadoras de poros altamente hemolíticas con una masa molecular de alrededor de 20 kDa y alta afinidad por las membranas que contienen esfingomielina. Sobre el extracto crudo de la anémona de mar Anthopleura nigrescens, se detectó la actividad hemolítica (HA). Para identificar la presencia de proteínas formadoras de poros similares a las actinoporinas en esta anémona, se realizó el fraccionamiento y análisis de su extracto crudo. El extracto acuoso de todo el cuerpo se sometió a cromatografía de filtración en gel en medio Sephadex G50 dando tres picos resueltos (P-I, P-II y P-III) medidos por su absorbancia a 280 nm. La caracterización funcional del extracto crudo y las fracciones cromatográficas se evaluó mediante HA frente a glóbulos rojos humanos. El extracto crudo y dos picos (P-II y P-III) mostraron HA. Curiosamente, el HA del extracto crudo y P-II fueron inhibidos específicamente por pequeñas vesículas unilaminares de fosfatidilcolina: esfingomielina (1: 1). Tanto el extracto crudo como el P-II revelaron la existencia de al menos una banda de proteína alrededor de 20 kDa por SDS-PAGE. La inhibición por esfingomielina del extracto de HA de todo el cuerpo y la localización de esta propiedad en P-II que podría estar asociada con una proteína de alrededor de 20 kDa sugieren la presencia de al menos una actinoporina nueva en A. nigrescens. Además, este es el primer informe de una actividad bioquímica de esta anémona de mar. Se está trabajando para purificar y caracterizar las entidades moleculares responsables de este HA inhibido por la esfingomielina.Universidad Nacional, Costa RicaEscuela de Ciencias Biológica

The Important Role of Membrane Fluidity on the Lytic Mechanism of the α-Pore-Forming Toxin Sticholysin I

Actinoporins have emerged as archetypal α-pore-forming toxins (PFTs) that promote the formation of pores in membranes upon oligomerization and insertion of an α-helix pore-forming domain in the bilayer. These proteins have been used as active components of immunotoxins, therefore, understanding their lytic mechanism is crucial for developing this and other applications. However, the mechanism of how the biophysical properties of the membrane modulate the properties of pores generated by actinoporins remains unclear. Here we studied the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs. To modulate membrane fluidity we used vesicles made of an equimolar mixture of phosphatidylcholine (PC) and egg sphingomyelin (eggSM), in which PC contained fatty acids of different acyl chain lengths and degrees of unsaturation. Our detailed single-vesicle analysis revealed that when membrane fluidity is high, most of the vesicles are partially permeabilized in a graded manner. In contrast, more rigid membranes can be either completely permeabilized or not, indicating an all-or-none mechanism. Altogether, our results reveal that St I pores can be heterogeneous in size and stability, and that these properties depend on the fluid state of the lipid bilayer. We propose that membrane fluidity at different regions of cellular membranes is a key factor to modulate the activity of the actinoporins, which has implications for the design of different therapeutic strategies based on their lytic action

Ferroptotic pores induce Ca2+ fluxes and ESCRT-III activation to modulate cell death kinetics

Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca2+ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of a few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca2+ fluxes during ferroptosis induce the activation of the ESCRT-III-dependent membrane repair machinery, which counterbalances the kinetics of cell death and modulates the immunological signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained increase of cytosolic Ca2+ prior to plasma membrane rupture is a common feature of regulated types of necrosis and position ESCRT-III activation as a general protective mechanism in these lytic cell death pathways

The presence of sterols favors Sticholysin I - membrane association and pore formation regardless of their ability to form laterally segregated domains

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT. As for actinoporins, it has been proposed that the presence of cholesterol (Chol) and the coexistence of lipid phases increase binding to the target membrane and pore-forming ability. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, and the presence of lipid domains) on the activity of actinoporins or which regions of the membrane are the most favorable for protein insertion, oligomerization, and eventually pore formation. To gain insight into the role of membrane properties on the functional activity of St I, we studied its binding to monolayers and vesicles of phosphatidylcholine (PC), sphingomyelin (SM), and sterols inducing (ergosterol -Erg and cholesterol -Chol) or not (cholestenone - Cln) membrane phase segregation in liquid ordered (Lo) and liquid disordered (Ld) domains. This study revealed that St I binds and permeabilizes with higher efficiency sterol-containing membranes independently of their ability to form domains. We discuss the results in terms of the relevance of different membrane properties for the actinoporins mechanism of action, namely, molecular heterogeneity, specially potentiated in membranes with sterols inducers of phase separation (Chol or Erg) or Cln, a sterol noninducer of phase separation but with a high propensity to induce nonlamellar phase. The role of the Ld phase is pointed out as the most suitable platform for pore formation. In this regard, such regions in Chol-containing membranes seem to be the most favored due to its increased fluidity; this property promotes toxin insertion, diffusion, and oligomerization leading to pore formation.Fil: Pedrera Puentes, Lohans. Universidad de La Habana; CubaFil: Gomide, Andreza. B.. Universidade de Sao Paulo; BrasilFil: Sanchez, Rafael E.. Universidad de La Habana; CubaFil: Ros Quincoces, Uris. Universidad de La Habana; CubaFil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Pazos, Fabiola. Universidad de La Habana; CubaFil: Lanio, María E.. Universidad de La Habana; CubaFil: Itri, Rosangela. Universidade de Sao Paulo; BrasilFil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Álvarez Valcárcel, Carlos Manuel. Universidad de La Habana; Cub