During animal fasting, the nutrient supply and metabolism switch from carbohydrates to a new reliance on the catabolism of energy‐dense lipid stores. Assembled under tight regulation, βγ‐CAT (a ...complex of non‐lens βγ‐crystallin and trefoil factor) is a pore‐forming protein and trefoil factor complex identified in toad Bombina maxima. Here, we determined that this protein complex is a constitutive component in toad blood, that actively responds to the animal fasting. The protein complex was able to promote cellular albumin and albumin‐bound fatty acid (FA) uptake in a variety of epithelial and endothelial cells, and the effects were attenuated by a macropinocytosis inhibitor. Endothelial cell‐derived exosomes containing largely enriched albumin and FAs, called nutrisomes, were released in the presence of βγ‐CAT. These specific nutrient vesicles were readily taken up by starved myoblast cells to support their survival. The results uncovered that pore‐forming protein βγ‐CAT is a fasting responsive element able to drive cell vesicular import and export of macromolecular nutrients.
Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to ...cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual‐color single‐molecule localization‐based super‐resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc‐shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.
Synopsis
Super‐resolution analyses show that Bax oligomerizes into lines, arcs, and rings to mediate mitochondrial outer membrane permeabilization during apoptosis.
Active Bax oligomerizes into full rings, lines, and arcs in apoptotic mitochondria.
Rings and arc‐shaped Bax assemblies are able to partially or completely line pores in lipid membranes.
Super‐resolution analyses show that Bax oligomerizes into lines, arcs, and rings to mediate mitochondrial outer membrane permeabilization during apoptosis.
Pyroptosis was long regarded as caspase-1-mediated monocyte death in response to certain bacterial insults. Caspase-1 is activated upon various infectious and immunological challenges through ...different inflammasomes. The discovery of caspase-11/4/5 function in sensing intracellular lipopolysaccharide expands the spectrum of pyroptosis mediators and also reveals that pyroptosis is not cell type specific. Recent studies identified the pyroptosis executioner, gasdermin D (GSDMD), a substrate of both caspase-1 and caspase-11/4/5. GSDMD represents a large gasdermin family bearing a novel membrane pore-forming activity. Thus, pyroptosis is redefined as gasdermin-mediated programmed necrosis. Gasdermins are associated with various genetic diseases, but their cellular function and mechanism of activation (except for GSDMD) are unknown. The gasdermin family suggests a new area of research on pyroptosis function in immunity, disease, and beyond.
The necrotic nature of pyroptosis was not well appreciated for decades and it was misregarded as a special type of apoptosis in monocytes due to the involvement of a caspase (caspase-1).
Characterization of inflammasomes establishes caspase-1-mediated pyroptosis as a general innate immune effector mechanism.
Caspase-4, 5, and 11, expressed also in nonmonocytic cells, induce pyroptosis upon recognition of intracellular lipopolysaccharide. The role of caspase-11 in endotoxic shock emphasizes the physiological importance of pyroptosis.
Both caspase-1 and caspase-11/4/5 cleave gasdermin D (GSDMD), a gasdermin-family member, to release its gasdermin-N domain that perforates the plasma membrane to induce cell swelling and osmotic lysis.
Nearly all gasdermins share the pore-forming and pyroptotic activity of GSDMD. Several gasdermins are associated with genetic diseases but their function and activation mechanism are unknown.
The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How ...the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a β sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the β sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.
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•Site-specific autoprocessing of caspase-4/11 allows high-affinity binding to GSDMD•Structures of caspase-4/11-GSDMD complexes reveal a substrate-binding exosite•Cleavage of GSDMD relies on the exosite interaction but not tetrapeptide sequence•The caspase-1-GSDMD complex structure shows the same GSDMD-targeting mechanism
Structural information provides insights into how site-specific processing of caspases allows the cleavage of the pore-forming protein gasdermin D to result in pyroptosis.
Perforin, a pore‐forming glycoprotein, has been demonstrated to play key roles in clearing virus‐infected cells and tumour cells due to its ability of forming ‘pores’ on the cell membranes. ...Additionally, perforin is also found to be associated with human diseases such as tumours, virus infections, immune rejection and some autoimmune diseases. Until now, plenty of perforin genes have been identified in vertebrates, especially the mammals and teleost fish. Conversely, vertebrate homologue of perforin gene was not identified in the invertebrates. Although recently there have been several reviews focusing on perforin and granzymes in mammals, no one highlighted the current advances of perforin in the other vertebrates. Here, in addition to mammalian perforin, the structure, evolution, tissue distribution and function of perforin in bony fish are summarized, respectively, which will allow us to gain more insights into the perforin in lower animals and the evolution of this important pore‐forming protein across vertebrates.
Perforin‐2 (PFN2, MPEG1) is a key pore‐forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane‐bound pre‐pore complex that converts to a ...pore‐forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo‐electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre‐pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre‐assembled complete pre‐pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre‐pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β‐hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre‐pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.
Synopsis
Perforin‐2 is an innate immune effector protein that forms pores in the membranes of phagocytosed bacteria. Here, cryo‐EM imaging of pre‐pore and pore complexes of perforin‐2 in isolation and after self‐assembly on membranes reveal new insights into membrane‐binding and pore formation.
Pre‐pore complexes formed on membranes bind in a distinct conformation to those observed when complexes form in isolation, before membrane binding.
Two types of pore structure can assemble: a closed flat ring and a twisted form that has the capacity to distort membrane bilayers.
Pores formed on membranes are mostly open arcs of subunits rather than closed rings.
The twisted pore conformation suggests a mechanism enabling a 180° rotation of the pore‐forming domain during membrane insertion and leading to clockwise propagation of subunit conformational change from pre‐pore to pore states.
Cryo‐EM imaging of the bactericidal innate immunity protein perforin‐2 reveals a large rotational conformational change that enables membrane insertion during pore formation.
Molecular mechanism of pore formation by aerolysin-like proteins Podobnik, Marjetka; Kisovec, Matic; Anderluh, Gregor
Philosophical transactions of the Royal Society of London. Series B. Biological sciences,
08/2017, Letnik:
372, Številka:
1726
Journal Article
Recenzirano
Odprti dostop
Aerolysin-like pore-forming proteins are an important family of proteins able to efficiently damage membranes of target cells by forming transmembrane pores. They are characterized by a unique domain ...organization and mechanism of action that involves extensive conformational rearrangements. Although structures of soluble forms of many different members of this family are well understood, the structures of pores and their mechanism of assembly have been described only recently. The pores are characterized by well-defined β-barrels, which are devoid of any vestibular regions commonly found in other protein pores. Many members of this family are bacterial toxins; therefore, structural details of their transmembrane pores, as well as the mechanism of pore formation, are an important base for future drug design. Stability of pores and other properties, such as specificity for some cell surface molecules, make this family of proteins a useful set of molecular tools for molecular recognition and sensing in cell biology.
This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.
BAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains ...obscure. Here, we report that BAX and BAK present distinct oligomerization properties, with BAK organizing into smaller structures with faster kinetics than BAX. BAK recruits and accelerates BAX assembly into oligomers that continue to grow during apoptosis. As a result, BAX and BAK regulate each other as they co-assemble into the same apoptotic pores, which we visualize. The relative availability of BAX and BAK molecules thereby determines the growth rate of the apoptotic pore and the relative kinetics by which mitochondrial contents, most notably mtDNA, are released. This feature of BAX and BAK results in distinct activation kinetics of the cGAS/STING pathway with implications for mtDNA-mediated paracrine inflammatory signaling.
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•BAK oligomerizes faster into smaller lines, arcs, and rings than those formed by BAX•BAK recruits BAX to co-assemble into the same supra-molecular apoptotic structures•BAX and BAK reciprocal regulation defines the growth dynamics of apoptotic pores•BAX and BAK have distinct roles in modulating the inflammatory outcome of apoptosis
BAX and BAK mediate membrane permeabilization in the mitochondrial pathway of apoptosis. Cosentino et al. show that the growth rate and permissiveness to mtDNA of the apoptotic pore can be dynamically modulated by the balance between BAX and BAK molecules, which regulate each other’s assembly, thereby impacting downstream inflammatory signaling.
Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in ...immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.
Various aerolysin‐like pore‐forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains ...unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin‐like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β‐prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high‐mannose glycans triggers drastic conformational changes of the aerolysin module in a pH‐dependent manner, ultimately resulting in the formation of a membrane‐bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore‐forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish‐specific defense molecule.
Synopsis
This study presents the first structures of a vertebrate aerolysin‐like pore‐forming protein, Dln1, as a water‐soluble dimer and membrane‐bound octameric quasi‐pore. Pore formation involves a conformational change, which might be triggered by binding of the lectin module of Dln1 to mannan.
The structures of the water‐soluble Dln1 dimer and membrane‐bound octameric quasi‐pore were determined by X‐ray crystallography and electron microscopy, respectively.
The pore‐forming process by Dln1 is activated by a mechanism distinct from the previously characterized bacterial members.
Dln1 and its homologs might function as novel defense molecules and are conserved in bony fishes and lamprey.
This study presents the first structures of a vertebrate aerolysin‐like pore‐forming protein, Dln1, as a water‐soluble dimer and membrane‐bound octameric quasi‐pore. Pore formation involves a conformational change, which might be triggered by binding of the lectin module of Dln1 to mannan.
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