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Fc region binding peptide conjugated with attenuated cationic amphiphilic lytic peptide L17E trimer [FcB(L17E)₃] was designed for immunoglobulin G (IgG) delivery into cells. Particle-like liquid droplets were generated by mixing Alexa Fluor 488 labeled IgG (Alexa488-IgG) with FcB(L17E)₃. Droplet contact with the cellular membrane led to spontaneous influx and distribution of Alexa488-IgG throughout cells in serum containing medium. Involvement of cellular machinery accompanied by actin polymerization and membrane ruffling was suggested for the translocation. Alexa488-IgG negative charges were crucial in liquid droplet formation with positively charged FcB(L17E)₃. Binding of IgG to FcB(L17E)₃ may not be necessary. Successful intracellular delivery of Alexa Fluor 594-labeled anti-nuclear pore complex antibody and anti-mCherry-nanobody tagged with supernegatively charged green fluorescence protein allowed binding to cellular targets in the presence of FcB(L17E)₃., 抗体を液滴に濃縮し細胞内へ高速輸送 --クモ毒改良ペプチドと抗体による液－液相分離の誘起と抗体の細胞内輸送--. 京都大学プレスリリース. 2021-08-06.

Pore forming toxins (PFTs) are the largest class of bacterial toxins playing a central role in bacterial pathogenesis. They are proteins specifically designed to form nanochannels in the membranes of target cells, ultimately resulting in cell death and establishing infection. PFTs are broadly classified as α- and β-PFTs, depending on secondary structures that form the transmembrane channel. A unique feature about this class of proteins is the drastic conformational changes and complex oligomerization pathways that occur upon exposure to the plasma membrane. A molecular understanding of pore formation has implications in designing novel intervention strategies to combat rising antimicrobial resistance, targeted-cancer therapy, as well as designing nanopores for specialized technologies. Central to unraveling the pore formation pathway is the availability of high resolution crystal structures. In this regard, β-toxins are better understood, when compared with α-toxins whose pore forming mechanisms are complicated by an incomplete knowledge of the driving forces for amphiphatic membrane-inserted helices to organize into functional pores. With the publication of the first crystal structure for an α-toxin, cytolysin A (ClyA), in 2009 we embarked on an extensive multiscale study to unravel its pore forming mechanism. This Account represents the collective mechanistic knowledge gained in our laboratories using a variety of experimental and theoretical techniques which include large scale molecular dynamics (MD) simulations, kinetic modeling studies, single-molecule fluorescence imaging, and super-resolution spectroscopy. We reported MD simulations of the ClyA protomer, oligomeric intermediates, and full pore complex in a lipid bilayer and mapped the conformational transitions that accompany membrane binding. Using single-molecule fluorescence imaging, the conformational transition was experimentally verified by analysis of various diffusion states of membrane bound ClyA. Importantly, we have uncovered a hitherto unknown putative cholesterol binding motif in the membrane-inserted helix of ClyA. Distinct binding pockets for cholesterol formed by adjacent membrane-inserted helices are revealed in MD simulations. Cholesterol appears to play a dual role by stabilizing both the membrane-inserted protomer as well as oligomeric intermediates. Molecular dynamics simulations and kinetic modeling studies suggest that the membrane-inserted arcs oligomerize reversibly to form the predominant transmembrane oligomeric intermediates during pore formation. We posit that this mechanistic understanding of the complex action of α-PFTs has implications in unraveling pore assembly across the wider family of bacterial toxins. With emerging antimicrobial resistance, alternate therapies may rely on disrupting pore functionality or oligomerization of these pathogenic determinants utilized by bacteria, and our study includes assessing the potential for dendrimers as pore blockers.

Pore forming proteins are a broad class of pathogenic proteins secreted by organisms as virulence factors due to their ability to form pores on the target cell membrane. Bacterial pore forming toxins (PFTs) belong to a subclass of pore forming proteins widely implicated in bacterial infections. Although the action of PFTs on target cells have been widely investigated, the underlying membrane response of lipids during membrane binding and pore formation has received less attention. With the advent of superresolution microscopy as well as the ability to carry out molecular dynamics (MD) simulations of the large protein membrane assemblies, novel microscopic insights on the pore forming mechanism have emerged over the last decade. In this review, we focus primarily on results collated in our laboratory which probe dynamic lipid reorganization induced in the plasma membrane during various stages of pore formation by two archetypal bacterial PFTs, cytolysin A (ClyA), an α-toxin and listeriolysin O (LLO), a β-toxin. The extent of lipid perturbation is dependent on both the secondary structure of the membrane inserted motifs of pore complex as well as the topological variations of the pore complex. Using confocal and superresolution stimulated emission depletion (STED) fluorescence correlation spectroscopy (FCS) and MD simulations, lipid diffusion, cholesterol reorganization and deviations from Brownian diffusion are correlated with the oligomeric state of the membrane bound protein as well as the underlying membrane composition. Deviations from free diffusion are typically observed at length scales below ∼130 nm to reveal the presence of local dynamical heterogeneities that emerge at the nanoscale—driven in part by preferential protein binding to cholesterol and domains present in the lipid membrane. Interrogating the lipid dynamics at the nanoscale allows us further differentiate between binding and pore formation of β- and α-PFTs to specific domains in the membrane. The molecular insights gained from the intricate coupling that occurs between proteins and membrane lipids and receptors during pore formation are expected to improve our understanding of the virulent action of PFTs.

Significance Synthetic nanomaterials are being sought to shuttle therapeutic payloads directly into the cell nucleus as a major target for chemo- and gene-based therapies. However, it remains uncertain whether and how synthetic entities are able to bypass the nuclear pore complexes (NPCs) that regulate transport into and out of the nucleus. We have constructed biocompatible polymer vesicles that infiltrate NPCs and resolved their nuclear uptake mechanism in vitro and in vivo. Their ability to deliver payloads directly into cell nuclei is further validated by transmission electron microscopy., Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.

Science advances 7(26), eabg9923 - (2021). doi:10.1126/sciadv.abg9923, The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries., Published by Assoc., Washington, DC [u.a.]

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, CatSper’s complex subunit composition has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

Summary: Preclinical models of ischemia/reperfusion injury (RI) demonstrate the deleterious effects of permeability transition pore complex (PTPC) opening in the first minutes upon revascularization of the occluded vessel. The ATP synthase c subunit (Csub) influences PTPC activity in cells, thus impacting tissue injury. A conserved glycine-rich domain in Csub is classified as critical because, when mutated, it modifies ATP synthase properties, protein interaction with the mitochondrial calcium (Ca2+) uniporter complex, and the conductance of the PTPC. Here, we document the role of a naturally occurring mutation in the Csub-encoding ATP5G1 gene at the G87 position found in two ST-segment elevation myocardial infarction (STEMI) patients and how PTPC opening is related to RI in patients affected by the same disease. We report a link between the expression of ATP5G1G87E and the response to hypoxia/reoxygenation of human cardiomyocytes, which worsen when compared to those expressing the wild-type protein, and a positive correlation between PTPC and RI.

Iota toxin, a type of A-B toxin produced by Clostridium perfringens, comprises an enzymatic component (Ia) and a membrane-binding component (Ib). The translocation of Ia to the target cell via the pore formed by Ib allows it to function as an ADP-ribosyltransferase that inhibits actin polymerization in the host cell. The structure of Ia-bound Ib-pore has been determined using cryo-electron microscopy (cryo-EM), thereby elucidating the mechanism of the initial Ia translocation; however, open questions regarding Ia translocation still exist. In this chapter, we describe a new method of preparing Ia-bound Ib-pore complex samples for structural analysis at high resolution using cryo-EM. This method is different from previously reported methods for other A-B toxins. Consequently, it produces Ib-pore with two different states with short and long membrane-spanning β-barrel stem. We expect that this method will be useful in functional and structural studies of iota toxin and other binary toxins.

To establish an infection, pathogenic mycobacteria use the Type VII secretion or ESX system to secrete virulence proteins across their cell envelope. The five ESX systems (ESX-1 to ESX-5) have evolved diverse functions in the cell, with the ESX-5 found almost exclusively in pathogens. Here we present a high-resolution cryo-electron microscopy structure of the hexameric ESX-5 Type VII secretion system. This 2.1 MDa membrane protein complex is built by a total of 30 subunits from six protomeric units, which are composed of the core components EccB5, EccC5, two copies of EccD5, and EccE5. The hexameric assembly of the overall ESX-5 complex is defined by specific inter-protomer interactions mediated by EccB5 and EccC5. The central transmembrane pore is formed by six pairs of EccC5 transmembrane helices that adopt a closed conformation in the absence of substrate in our structure. On the periplasmic face of the ESX-5 complex, we observe an extended arrangement of the six EccB5 subunits around a central cleft. Our structural findings provide molecular details of ESX-5 assembly and observations of the central secretion pore, which reveal insights into possible gating mechanisms used to regulate the transport of substrates.

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

Parasitic protozoans of the genus Theileria are intracellular pathogens that induce the cellular transformation of leukocytes, causing uncontrolled proliferation of the infected host cell. The transforming stage of the parasite has a strictly intracellular lifestyle and ensures its distribution to both daughter cells during host cell cytokinesis by aligning itself across the metaphase plate and by binding tightly to central spindle and astral microtubules. Given the importance of the parasite surface in maintaining interactions with host microtubules, we analyzed the ultrastructure of the host-parasite interface using transmission electron microscopy combined with high-resolution fluorescence microscopy and live-cell imaging. We show that porous membranes, termed annulate lamellae (AL), closely associate with the Theileria surface in infected T cells, B cells, and macrophages and are not detectable in noninfected bovine cell lines such as BL20 or BoMACs. AL are membranous structures found in the cytoplasm of fast-proliferating cells such as cancer cells, oocytes, and embryonic cells. Although AL were first observed more than 60 years ago, the function of these organelles is still not known. Indirect immunofluorescence analysis with a pan-nuclear pore complex antibody, combined with overexpression of a panel of nuclear pore proteins, revealed that the parasite recruits nuclear pore complex components close to its surface. Importantly, we show that, in addition to structural components of the nuclear pore complex, nuclear trafficking machinery, including importin beta 1, RanGAP1, and the small GTPase Ran, also accumulated close to the parasite surface. IMPORTANCETheileria schizonts are the only known eukaryotic organisms capable of transforming another eukaryotic cell; as such, probing of the interactions that occur at the host-parasite interface is likely to lead to novel insights into the cell biology underlying leukocyte proliferation and transformation. Little is known about how the parasite communicates with its host or by what route secreted parasite proteins are translocated into the host, and we propose that nuclear trafficking machinery at the parasite surface might play a role in this. The function of AL remains completely unknown, and our work provides a basis for further investigation into the contribution that these porous, cytomembranous structures might make to the survival of fast-growing transformed cells.

In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains αA-β1-β2 and β3-β4-αB-β5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first α-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.

Spreading depolarization is a wave of neuronal and glial depolarization. Within minutes after spreading depolarization, the neuronal hemichannel pannexin 1 (PANX1) opens and forms a pore complex with the ligand-gated cation channel P2X7, allowing the release of excitatory neurotransmitters to sustain spreading depolarization and activate neuroinflammation. Here, we explore the hypothesis that the P2X7–PANX1 pore complex is a critical determinant of spreading depolarization susceptibility with important consequences for neuroinflammation and trigeminovascular activation. We found that genetic loss of function or ablation of the P2x7 gene inhibits spreading depolarization. Moreover, pharmacological suppression of the P2X7–PANX1 pore complex inhibits spreading depolarization in mice carrying the human familial hemiplegic migraine type 1 R192Q missense mutation as well as in wild-type mice and rats. Pore inhibitors elevate the electrical threshold for spreading depolarization, and reduce spreading depolarization frequency and amplitude. Pore inhibitors also suppress downstream consequences of spreading depolarization such as upregulation of interleukin-1 beta, inducible nitric oxide synthase and cyclooxygenase-2 in the cortex after spreading depolarization. In addition, they inhibit surrogates for trigeminovascular activation, including expression of calcitonin gene-related peptide in the trigeminal ganglion and c-Fos in the trigeminal nucleus caudalis. Our results are consistent with the hypothesis that the P2X7–PANX1 pore complex is a critical determinant of spreading depolarization susceptibility and its downstream consequences, of potential relevance to its signature disorders such as migraine.

Infections in many virulent bacterial strains are triggered by the release of pore forming toxins (PFTs), which form oligomeric transmembrane pore complexes on the target plasma membrane. The spatial extent of the perturbation to the surrounding lipids during pore formation is relatively unexplored. Using all-atom molecular dynamics simulations, we investigate the changes in the structure and dynamics of lipids in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) lipid bilayer in the presence of contrasting PFTs. Cytolysin A (ClyA) anαtoxin with its inserted wedge shaped bundle of insertedαhelices induces significant asymmetry across the membrane leaflets in comparison withαhemolysin (AHL) aβtoxin. Despite the differences in hydrophobic mismatch and uniquely different topologies of the two oligomers, perturbation to lipid order as reflected in the tilt angle and order parameters, and membrane thinning is short ranged, lying within ∼ 2.5 nm from the periphery of the either pore complex, commensurate with distances typically associated with van der Waals forces. In contrast, the spatial extent of perturbations to the lipid dynamics extend outward to at least 4 nm for both proteins, and the continuous survival probabilities reveal the presence of a tightly bound shell of lipids in this region. Displacement probability distributions show long tails and the distinctly non-Gaussian features reflect the induced dynamic heterogeneity. A detailed profiling of the protein-lipid contacts with residues tyrosine, tryptophan, lysine and arginine show increased non-polar contacts in the cytoplasmic leaflet for both PFTs, with a higher number of atomic contacts in the case of AHL in the extracellular leaflet due to the mushroom-like topology of the pore complex. The short ranged nature of the perturbations observed in this simple one component membrane suggests an inherent plasticity of membrane lipids enabling recovery of structure and membrane fluidity even in the presence of these large oligomeric trans-membrane protein assemblies. This observation has implications in membrane repair processes such as budding or vesicle fusion events used to mitigate PFT virulence, where the underlying lipid dynamics and fluidity in the vicinity of the pore complex are expected to play an important role.

Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.

To explore the involvement of protein lysine acetylation in the conversion of muscle to meat, a quantitative analysis of the acetylome in postmortem porcine muscle with or without antemortem stress was conducted. In total, 771 acetylpeptides containing 681 lysine acetylation sites mapping to 176 acetylproteins were identified. Acetylproteins were enriched in muscle contraction, carbohydrate metabolism, cell apoptosis and calcium signaling. Bioinformatic analysis suggests that preslaughter handling may be associated with glycolysis in postmortem muscle and the overall meat quality, via acetylation of multiple enzymes of glycogenolysis/glycolysis, regulate rigor mortis via acetylation of contractile, ATP production and calcium signaling-related proteins, and regulate stress response, cell apoptosis and meat tenderization via regulating the functions of heat shock proteins and permeability transition pore complex. This study provides the first overview of the acetylome in postmortem muscle as affected by preslaughter handling and broadens knowledge of the biochemistry regulating meat quality development.

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery—also named injectisome—assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors., Author summary Many human, animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells. Because injectisomes are crucial for these bacteria’s infectious potential they have been considered as targets for antiinfective drugs. Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level. However, only little information is available for a central part of the injectisome named the translocon. This pore-like assembly integrates into host cell membranes and thereby serves as an entry gate for the bacterial toxins. We used state of the art fluorescence microscopy to watch translocons of the diarrheagenic pathogen Yersinia enterocolitica during infection of human host cells. Thereby we could for the first time—with fluorescence microscopy—visualize translocons connected to other parts of the injectisome. Furthermore, because translocons mark functional injectisomes we could obtain evidence that injectisomes only become active for secretion of translocators when the bacteria are almost completely enclosed by host cells. These findings provide a novel view on the organization and regulation of bacterial translocons and may thus open up new strategies to block the function of infectious bacteria.

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery - also named injectisome - assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their assembly have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualizeY. enterocoliticatranslocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the putatively operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology we provide novel information about the spatiotemporal organisation of T3SS translocons and their regulation by host cell factors.Author SummaryMany human, animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells. Because injectisomes are crucial for these bacteria’s infectious potential they have been considered as targets for antiinfective drugs. Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level. However, only little information is available for a central part of the injectisome named the translocon. This pore-like assembly integrates into host cell membranes and thereby serves as an entry gate for the bacterial toxins. We used state of the art fluorescence microscopy to watch translocons of the diarrheagenic pathogenYersinia enterocoliticaduring infection of human host cells. Thereby we could for the first time - with fluorescence microscopy - visualize translocons connected to other parts of the injectisome. Furthermore, because translocons mark functional injectisomes we could obtain evidence that injectisomes only become active when the bacteria are almost completely enclosed by host cells. These findings provide a novel view on the organisation and regulation of bacterial translocons and may thus open up new strategies to block the function of infectious bacteria.

eLife 7, e38017 (2018). doi:10.7554/eLife.38017, α-Xenorhabdolysins (Xax) are α-pore-forming toxins (α-PFT) that form 1–1.3 MDa large pore complexes to perforate the host cell membrane. PFTs are used by a variety of bacterial pathogens to attack host cells. Due to the lack of structural information, the molecular mechanism of action of Xax toxins is poorly understood. Here, we report the cryo-EM structure of the XaxAB pore complex from Xenorhabdus nematophila and the crystal structures of the soluble monomers of XaxA and XaxB. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12–15 subunits containing pore, classifying XaxAB as bi-component α-PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. Based on our results, we propose a novel structural model for the mechanism of Xax intoxication., Published by eLife Sciences Publications, Cambridge

The mitochondrial permeability transition (MPT) consists of an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes, resulting in the osmotic breakout of the organelle. MPT drives cell death and provides an etiological contribution to several human disorders characterized by the acute loss of post-mitotic cells. These conditions include ischemia/reperfusion injury, cancer and neurodegenerative disorders. However, precise knowledge of the structure and regulators of the supramolecular entity that induces MPT, the so-called permeability transition pore complex (PTPC), is lacking and this constitutes a substantial obstacle in the development of MPT-targeting agents with clinical applications. Here we report the current evidences about molecular structure and regulatory components of PTPC. In particular we pay attention on new two proteins which recently were added to the list of PTPC components: the mitochondrial F1FO ATP synthase, particularly and the SPG7 paraplegin matrix AAA peptidase subunit. At least a detailed overview of MPT contribution to pathological condition is provided, focusing on the idea that to develop therapeutic drugs, it will be fundamental to understand the molecular composition of the PTPC.

Protein membrane interactions play an important role in our understanding of diverse phenomena ranging from membrane-assisted protein aggregation to oligomerization and folding. Pore-forming toxins (PFTs) are the primary vehicle for infection by several strains of bacteria. These proteins which are expressed in a water-soluble form (monomers) bind to the target membrane and conformationally transform (protomers) and self-assemble to form a multimer transmembrane pore complex through a process of oligomerization. On the basis of the structure of the transmembrane domains, PFTs are broadly classified into beta or alpha toxins. In contrast to beta-PFTs, the paucity of available crystal structures coupled with the amphipathic nature of the transmembrane domains has hindered our understanding of alpha-PFT pore formation. In this article, we use molecular dynamics (MD) simulations to examine the process of pore formation of the bacterial alpha-PFT, cytolysin A from Escherichia coli (ClyA) in lipid bilayer membranes. Using atomistic MD simulations ranging from 50 to 500 ns, we show that transmembrane oligomeric intermediates or ``arcs'' form stable proteolipidic complexes consisting of protein arcs with toroidal lipids lining the free edges. By creating initial conditions where the lipids are contained within the arcs, we study the dynamics of spontaneous lipid evacuation and toroidal edge formation. This process occurs on the time scale of tens of nanoseconds, suggesting that once protomers oligomerize, transmembrane arcs are rapidly stabilized to form functional water channels capable of leakage. Using umbrella sampling with a coarse-grained molecular model, we obtain the free energy of insertion of a single protomer into the membrane. A single inserted protomer has a stabilization free energy of -52.9 +/- 1.2 kJ/mol and forms a stable transmembrane water channel capable of leakage. Our simulations reveal that arcs are stable and viable intermediates that can occur during the pore-formation pathway for ClyA.

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.

The cytolytic toxin cytolysin A (ClyA) from Escherichia coli is probably one of the best-characterized examples of bacterial, α-pore–forming toxins (α-PFTs). Like other PFTs, ClyA exists in a soluble, monomeric form that assembles to an annular, homo-oligomeric pore complex upon contact with detergent or target membranes. Comparison of the three-dimensional structures of the 34 kDa monomer and the protomer in the context of the dodecameric pore complex revealed that ClyA undergoes one of the largest conformational transitions described for proteins so far, in which 55% of the residues change their position and 16% of the residues adopt a different secondary structure in the protomer. Studies on the assembly of ClyA revealed a unique mechanism that differs from the assembly mechanism of other PFTs. The rate-liming step of pore formation proved to be the unimolecular conversion of the monomer to an assembly-competent protomer, during which a molten globule-like off-pathway intermediate accumulates. The oligomerization of protomers to pore complexes is fast and follows a kinetic scheme in which mixtures of linear oligomers of different size are formed first, followed by very rapid and specific association of pairs of oligomers that can directly perform ring closure to the dodecameric pore complex. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane. DOI: http://dx.doi.org/10.7554/eLife.23644.001

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that assembles a type III secretion system (T3SS) on its surface. The last portion of the T3SS, called the 'translocon', is composed of a filament and a pore complex that is inserted into the membrane of intestinal epithelial cells. The genes encoding the translocon (espADB) are part of the LEE4 operon. Their expression is regulated by a complex post-transcriptional mechanism that involves the processing of LEE4 mRNA by the essential endoribonuclease RNase E. Here, we report the construction of an EHEC strain (TEA028-rne) in which RNase E can be induced by adding IPTG to the culture medium. EHEC cells deficient in RNase E displayed an abnormal morphology and slower growth, in agreement with published observations in E. coli K-12. Under those conditions, EspA and EspB were produced at higher concentrations, and protein secretion still occurred. These results indicate that RNase E negatively regulates translocon protein synthesis and demonstrate the utility of E. coli strain TEA028-rne as a tool for investigating the influence of this ribonuclease on EHEC gene expression in vitro.

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is a soluble, 34 kDa monomer that assembles into a dodecameric pore complex in the presence of membranes or detergent. The comparison of the X-ray structures of monomeric ClyA and the ClyA protomer in the pore complex revealed one of the largest conformational transitions observed so far in proteins, involving the structural rearrangement of more than half of all residues, which is consistent with the finding that conversion from the monomer to the assembly competent protomer is rate-limiting for pore assembly. Here, we introduced artificial disulfide bonds at two distinct sites into the ClyA monomer that both prevent a specific structural rearrangement required for protomer formation. Using electron microscopy and hemolytic activity assays, we show that the engineered disulfides indeed trap these ClyA variants in an assembly incompetent state. Assembly of the variants into functional pore complexes can be completely recovered by disulfide reduction. The assembly kinetics of the ClyA variants recorded with circular dichroism and fluorescence spectroscopy revealed the same mechanism of protomer formation that was observed for wild-type ClyA, proceeding via an intermediate with decreased secondary structure content.

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å β barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F 1 F O ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F 1 F O ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F 1 F O ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT. Specific mutations in the F 1 F O ATP synthase c subunit that alter C‐ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F 1 F O ATP synthase dimers. Destabilizing F 1 F O ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT. The current study does not provide direct evidence that the C‐ring is the long‐sought pore‐forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F 1 F O ATP synthase dimers and involves the C‐ring.

Despite the great potential of antibodies as intracellular therapeutics, there is a significant, unmet challenge in delivering sufficient amounts of folded antibodies inside cells. We describe an all-protein self-assembled nanocarrier capable of delivering functional antibodies to the cytosol. By combining an α-helical peptide that self-assembles into a hexameric coiled-coil bundle and an Fc-binding Protein A fragment, we generated the Hex nanocarrier that is efficiently internalized by cells without cytotoxicity. Localization of multiple Fc-binding domains on the hexameric core allowed the Hex nanocarrier to tightly bind antibody with sub-nanomolar affinity regardless of pH and the antibody's originating species. The size of the Hex nanocarrier ranges from 25 to 35nm depending on the antibody loading ratio. We demonstrated the capacity of the Hex nanocarrier to deliver functional antibodies to the cytosol by employing anti-β-tubulin or anti-nuclear pore complex antibody as cargo. The design of the Hex nanocarrier is modular, which enables functionalization beyond Fc-binding. We exploited this feature to improve the cytosolic delivery efficiency of the Hex nanocarrier by addition of an endosomolytic motif to the core. The modified Hex nanocarrier exhibited similar antibody-binding behavior, but delivered more antibodies to their cytosolic targets at a faster rate. This work demonstrates an efficient intracellular antibody delivery platform with significant advantages over existing approaches as it does not require modification of the antibody, is biodegradable, and has an antibody to carrier mass ratio of 13, which is greater than other reported antibody carriers.

Many important proteins function as multimeric complexes, which often contain different types and numbers of subunits. Such proteins play a key role in a wide variety of cellular events, including signal transduction, cell regulation, transport and energy generation. Protein oligomers are associated with many human diseases including early onset Parkinson’s, Alzheimer’s and Huntington’s disease. Such complexes also play key roles in the pathogenic action of many bacterial toxins. If we are to understand the widespread roles of protein complexes, additional methods must be developed that are capable of measuring the temporal variations in subunit stoichiometry that must occur during complex assembly. Several techniques provide information on protein stoichiometry. The mapping of high resolution X-ray crystal structures onto lower resolution cryoelectron microscopy images provides one method for determining high-resolution structures of large complexes. 6] The difficulty of this sophisticated approach, however, is reflected in the limited number of publications successfully combining both techniques. Atomic force microscopy (AFM) can also be used to determine subunit stoichiometry either directly or by specifically tagging subunits with other large molecules. Other techniques capable of providing information on the stoichiometry of protein complexes include analytical ultracentrifugation, gel electrophoresis, Fcrster resonance energy transfer (FRET) and mass spectrometry. These methods vary greatly in both experimental complexity and their utility for subunit determination. For example—although costly and time consuming—cryoelectron microscopy and X-ray crystallography are capable of providing exquisite resolution of large protein complexes. On the other hand, FRET only requires judicious chemical modification of two proteins, but is limited to studying the interactions of two (or possibly three) partners in what could be a much larger complex. Single-molecule fluorescence (SMF) microscopy is a relatively new technique capable of resolving the stoichiometry of a fluorescently labelled protein complex. SMF also has the potential to measure the dynamics of protein oligomerisation both in vitro and in vivo, without the need to immobilise the protein on a surface or within a crystal. SMF is not able to provide a structure of an individual molecule (as with AFM or cryoelectron microscopy), however, SMF can be used to visualise molecular complexes tagged with different fluorescent labels. One SMF method capable of resolving subunit stoichiometry is simply to count the number of photobleaching steps within a single complex. Photobleaching is the irreversible loss of fluorescence in a molecule due to changes in its structure following a light-induced chemical reaction. For an individual fluorophore, photobleaching is observed as a step change in fluorescence intensity. If each subunit within a protein complex is labelled with a fluorescent molecule, the total number of photobleaching steps determines the number of subunits within the complex. Multiple photobleaching steps have been observed in a randomly labelled mRNA–ribosome complex and in fluorescently labelled polymers. Photobleaching steps have also been used to determine the number of fluorescently labelled proteins encapsulated within a lipid vesicle, to infer the stoichiometry of stator components within the bacterial flagellar motor and to determine the number of labels on a green fluorescent protein dimer. Here, we employ the stepwise changes in SMF intensity during photobleaching to determine the number of subunits within a membrane–protein complex. b-Barrel pore-forming toxins (b-PFTs) are a class of multimeric membrane proteins that assemble on lipid membranes to form bilayer-spanning pores. Their known stoichiometries vary from small oligomers to large 30–50 subunit complexes. a-Hemolysin (aHL) is the archetypal b-barrel-forming toxin. High-resolution crystallographic, AFM and biochemical studies suggests that aHL forms a heptameric pore. Other AFM experiments have reported hexameric aHL, thus indicating that some variation in subunit stoichiometry might be possible. Leukocidin is a bicomponent b-PFT with significant sequence homology to aHL. It is formed from two different polypeptide subunits, LukF and LukS, both of which have several different isoforms. The structures of both LukS and LukF monomers have been resolved with X-ray crystallography. 30] However, in contrast to aHL, there is much less consensus regarding the structure of the fully assembled leukocidin pore. No crystal structure of the pore complex is available, and the numbers of each component are not conclusively known. Biochemical cross-linking and chemical modification during single-channel recording have provided evidence that leukocidin is an octameric pore with an alternating arrangement of subunits. Other experiments indicate that leukocidin forms pores with alternative stoichiometries: either hexameric or heptameric. Single-molecule FRET experiments have also been performed with leukocidin. Higuchi and co-workers [a] Dr. S. Cheley, Dr. M. I. Wallace, Prof. Dr. H. Bayley Department of Chemistry, University of Oxford Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+1)44-1865-285002 [b] Dr. S. K. Das, Dr. M. Darshi Department of Molecular & Cellular Medicine, The Texas A&M University System Health Science Center College Station, TX 77843-1114 (USA) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author: representative image showing individually resolved aHL complexes on a glass cover-slip surface, and a graph showing how the intensity of each single-molecule spot correlates with the number of photobleaching steps detected.

Despite numerous studies on utilizing polymeric vesicles as nanocapsules, fabrication of tunable molecular pathways on transportable vesicle walls remains challenging. Traditional methods for building penetrated channels on vesicular membrane surface often involve regulating the solvent polarity or photo-cross-linking. Herein, we developed a neat, green approach of stimulation by using CO2 gas as “molecular drill” to pierce macroporous structures on the membrane of polymersomes. By simply introducing CO2/N2 gases into the aqueous solution of self-assemblies without accumulating any byproducts, we observed two processes of polymeric shape transformation: “gas breathing” and “gas piercing.” Moreover, the pathways in terms of dimension and time were found to be adjustable simply by controlling the CO2 stimulation level for different functional encapsulated molecules in accumulation, transport, and releasing. CO2-breathing and piercing of polymersomes offers a promising functionality to tune nanocapsules for encapsulating and releasing fluorescent dyes and bioactive molecules in living systems and also a unique platform to mimic the structural formation of nucleus pore complex and the breathing process in human beings and animals.

Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.

Pore forming toxins (PFTs) are the primary vehicle for infection by several strains of bacteria. These proteins which are expressed in a water soluble form (monomers), bind to the target membrane and conformationally transform (protomers), and self-assemble to form a multimer transmembrane pore complex through a process of oligomerization. PFTs are broadly classified into or alpha or beta toxins. In contrast to beta-PFTs, the paucity of available crystal structures coupled with the amphipathic nature of the transmembrane domains have hindered our understanding of alpha-PFT pore formation. We use molecular dynamics (MD) simulations to examine the process of pore formation of the bacterial toxin Cytolysin A from Escherichia coli (ClyA) in lipid bilayer membranes. Using atomistic MD simulations ranging from 50- 500 ns, we show that transmembrane oligomeric intermediates or ‘arcs’ form stable proteo-lipidic complexes consisting of protein arcs with toroidal lipids lining the free edges. By creating initial conditions where the lipids are contained within the arcs, we study the dynamics of spontaneous lipid evacuation and toroidal edge formation. This process occurs on the timescale of tens of nanoseconds, suggesting that once protomers oligomerize, transmembrane arcs are rapidly stabilized to form functional water channels capable of leakage. Using umbrella sampling with a coarse grained molecular model, we obtain the free energy of insertion of a single protomer into the membrane. A single inserted protomer has a stabilization free energy of 53 kJ/mol and forms a stable transmembrane water channel capable of leakage. Our simulations reveal that arcs are stable and viable intermediates that can occur during the pore formation pathway for ClyA.

Triple A syndrome is a rare autosomal recessive disorder characterized by ACTH-resistant adrenal failure, alacrima, achalasia, and progressive neurological manifestations. The majority of cases are associated with mutations in the AAAS gene, which encodes a novel, 60-kDa WD-repeat nuclear pore protein, alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN) of unknown function. Our aim was to elucidate the functional role of ALADIN by determining its interacting protein partners using the bacterial two-hybrid (B2-H) technique. Nonidentical cDNA fragments were identified from both a HeLa S-3 cell and human cerebellar cDNA library that encoded the full-length ferritin heavy chain protein (FTH1). This interaction was confirmed by both co-immunoprecipitation and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. Immunoblotting showed that fibroblasts from triple A patients (with known AAAS mutations) lack nuclear FTH1, suggesting that the nuclear translocation of FTH1 is defective. Cells transfected with FTH1 and visualized by confocal microscopy had very little nuclear FTH1, but when cotransfected with AAAS, FTH1 is readily visible in the nuclei. Therefore, FTH1 nuclear translocation is enhanced when ALADIN is coexpressed in these cells. In addition to its well known iron storage role, FTH1 has been shown to protect the nucleus from oxidative damage. Apoptosis of neuronal cells induced by hydrogen peroxide was significantly reduced by transfection of AAAS or by FTH1 or maximally by both genes together. Taken together, this work offers a plausible mechanism for the progressive clinical features of triple A syndrome. (Molecular Endocrinology 23: 2086–2094, 2009)

Under physiological conditions, astrocytes take up L-glutamate from the synaptic gap, metabolize it to L-glutamine and return it to neurons, where L-glutamine is metabolized to L-glutamate and stored in neurotransmitter vesicles. However, under pathological conditions, such as hepatic failure, L-glutamine and ammonium are elevated globally in the brain. The Trojan horse hypothesis of L-glutamine toxicity assumes that intramitochondrial hydrolysis of L-glutamine enhances ammonium locally and leads to mitochondrial dysfunction. In the present study, we show that exposure of murine primary microglia as well as of the microglial cell-line BV-2 to L-glutamine promotes chromatin condensation and formation of crescent-like structures in the nucleus. Furthermore, L-glutamine induced an increase in annexin-V labelling, cell shrinkage (apoptotic volume decrease), cell fragmentation and formation of apoptotic bodies. Inhibition of the phosphate-activated glutaminase with 6-diazo-5-oxo-L-norleucine suppressed chromatin condensation and annexin-V labelling in L-glutamine-exposed cells. In addition, inhibition of the glutamine synthetase with L-methionine sulfoximine suppressed chromatin condensation and annexin-V labelling in ammonium-exposed cells. L-glutamine and ammonium enhanced production of reactive oxygen species, as detected with CM-H(2)DCFDA. Apoptosis, induced by L-glutamine, was inhibited either by the radical scavenger alpha-tocopherol or by the nitric oxide synthase blocker N (G)-methyl-L-arginine. Cyclosporin A, a ligand of the permeability transition pore complex component cyclophilin D, prevented L-glutamine-triggered apoptosis. Furthermore, blockade of caspase-9 activity with Z-LEHD-FMK prevented L-glutamine-triggered apoptosis. Taken together, our results indicate that hydrolysis of l-glutamine and, accordingly, accumulation of ammonium in mitochondria induce the intrinsic pathway of apoptosis, characterized by mitochondrial dysfunction and activation of caspase-9, which activates caspase-3.

This study shows the effects of the flavonoid quercetin on diverse mitochondrial functions, among them membrane permeability. Our findings indicate that the addition of 50 microM quercetin did not produce reactive oxygen derived species; however, it inhibited the oxidative stress induced after the addition of Fe(2)/H(2)O(2) by about 38%. At this concentration, quercetin also promoted a fast calcium release, inhibited oxidative phosphorylation, stimulated oxygen consumption, and decreased membrane potential. In addition 50 microM quercetin inhibited the adenine nucleotide translocase (ANT) by 46%. These effects induced the opening of the permeability transition pore and release of cytochrome c, by its interaction with a component of the non-specific pore complex, fixed to the carrier in the conformation c, as carboxyatractyloside does. Quercetin-induced permeability transition pore opening was inhibited by 0.5 microM cyclosporin A, but, interestingly, the release of cytochrome c was not inhibited by the immunosuppressor, as quercetin was found to disrupt the outer membrane.

Mutacin 1140 and nisin A are peptide antibiotics that belong to the lantibiotic family. N-Terminal rings A and B of nisin A and mutacin 1140 (lipid II-binding domain) share many structural and sequence similarities. Nisin A binds lipid II and thus disrupts cell wall synthesis and also forms transmembrane pores. Very little is known about mutacin 1140 in this regard. We performed fluorescence-based studies using a bacteria-mimetic membrane system. The results indicated that lipid II monomers are arranged differently in the mutacin 1140 complex than in the nisin A complex. These differences in complex formation may be attributed to the fact that nisin A uses lipid II to form a distinct pore complex, while mutacin 1140 does not form pores in this membrane system. Further experiments demonstrated that the mutacin 1140-lipid II and nisin A-lipid II complexes are very stable and capable of withstanding competition from each other. Transmembrane electrical potential experiments using a Streptococcus rattus strain, which is sensitive to mutacin 1140, demonstrated that mutacin 1140 does not form pores in this strain even at a concentration 8 times higher than the minimum inhibitory concentration (MIC). Circular complexes of mutacin 1140 and nisin A were observed by electron microscopy, providing direct evidence for a lateral assembly mechanism for these antibiotics. Mutacin 1140 did exhibit a membrane disruptive function in another commonly used artificial bacterial membrane system, and its disruptive activity was enhanced by increasing amounts of anionic phospholipids.

Nisin is the preeminent lantibiotic, and to date its antibacterial mechanism has been investigated using a variety of techniques. While nisin's lipid II-mediated mode of action is well-established, a detailed analysis of the thermodynamic parameters governing this interaction has not been previously reported. We here describe an approach employing isothermal titration calorimetry to directly measure the affinity of nisin for lipid II and a number of synthetic lipid II precursors and analogues. Our measurements confirm the pyrophosphate unit of lipid II as the primary site of nisin binding and also indicate that the complete MurNAc moiety is required for a high-affinity interaction. Additionally, we find that while the pentapeptide unit of the lipid II molecule is not required for strong binding by nisin, it does play an important role in stabilizing the subsequently formed nisin-lipid II pore complex, albeit at an entropic cost. The anchoring of lipid II in a membrane environment was also found to play a significant role in enhancing nisin binding and is required in order to achieve a high-affinity interaction.

Hypoxia stimulates hepatocellular carcinoma (HCC) cell growth via hexokinase (HK) II induction, and alternatively, HK II inhibition induces apoptosis by activating mitochondrial signaling. This study was to investigate whether the induction of HK II by hypoxia is associated with enhanced mitochondrial stability and to confirm the apoptosis-inducing efficacy of HK II inhibitor in an in vivo model of HCC. Mitochondrial stability was examined by treating isolated mitochondria with deoxycholate, a permeability-enhancing agent. Alteration of permeability transition pore complex composition was analyzed by immunoprecipitation and immunoblotting. An in vivo model of HCC was established in C3H mice i.d. implanted with MH134 cells. The antitumor efficacy of i.p. given 3-bromopyruvate (3-BrPA), a HK II inhibitor, was evaluated by measuring tumor volumes and quantifying apoptosis using terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining and 99mTc-hydrazinonicotinamide-Annexin V scans. Hypoxia enhanced mitochondrial stability, and this was inhibited by 3-BrPA treatment. In particular, HK II levels in permeability transition pore complex immunoprecipitates were reduced after 3-BrPA treatment. In mice treated with 3-BrPA, mean tumor volumes and tumor volume growth were found to be significantly reduced. Moreover, percentages of terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive cells were significantly increased in 3-BrPA–treated mice, and this apoptosis-inducing efficacy was reflected in vivo by 99mTc-hydrazinonicotinamide-Annexin V imaging. Our results show that hypoxia enhances mitochondrial stability via HK II induction and that HK II inhibitor treatment exhibits an in vivo antitumor effect by inducing apoptosis. Therefore, HK II inhibitors may be therapeutically useful for the treatment of advanced infiltrative hypovascular HCCs, which are growing in a hypoxic environment. [Mol Cancer Ther 2007;6(9):2554–62]

Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. This observation suggested that the D4 loops, which include loops L1–L3 and the undecapeptide, of ILY were no longer required for its cell binding. However, we show here that membrane insertion of the D4 loops is required for the cytolysis by ILY. Receptor binding triggers changes in the structure of ILY that are necessary for oligomerization, but membrane insertion of the D4 loops is critical for oligomer assembly and pore formation. Defects that prevent membrane insertion of the undecapeptide also block assembly of the prepore oligomer, while defects in the membrane insertion of the L1–L3 loops prevent the conversion of the prepore oligomer to the pore complex. These studies reveal that pore formation by ILY, and probably other CDCs, is affected by an intricate and coupled sequence of interactions between domain 4 and the membrane.

Perfringolysin O (PFO), a soluble toxin secreted by the pathogenic Clostridium perfringens, forms large homo-oligomeric pore complexes comprising up to 50 PFO molecules in cholesterol-containing membranes. In this study, electron microscopy (EM) and single-particle image analysis were used to reconstruct two-dimensional (2D) projection maps from images of oligomeric PFO prepore and pore complexes formed on cholesterol-rich lipid layers. The projection maps are characterized by an outer and an inner ring of density peaks. The outer rings of the prepore and pore complexes are very similar; however, the protein densities that make up the inner ring of the pore complex are more intense and discretely resolved than they are for the prepore complex. The change in inner-ring protein density is consistent with a mechanism in which the monomers within the prepore complex make a transition from a partially disordered state to a more ordered transmembrane beta-barrel in the pore complex. Finally, the orientation of the monomers within the oligomeric complexes was determined by visualization of streptavidin (SA) molecules bound to biotinylated cysteine-substituted residues predicted to face either the inner or outer surface of the oligomeric pore complex. This study provides an unprecedented view of the conversion of the PFO prepore to pore complex.

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

Under resting conditions, the endoplasmic reticulum (ER) intraluminal free calcium concentration ([Ca(2+)](ER)) reflects a balance between active uptake by Ca(2+)-ATPases and passive efflux via 'leak channels'. Despite their physiological importance and ubiquitous leak pathway mechanism, very little is known about the molecular nature of these channels. As it has been suggested that the open translocon pore complex of the ER is permeable to ions and neutral molecules, we hypothesized that the ribosome-bound translocon would be permeable to calcium after treatment with puromycin, a translation inhibitor that specifically releases polypeptide chains. At this time, the translocon channel is left open. We measured the fluctuations in cytoplasmic and luminal calcium concentrations using fluorescent dyes (fura-2 and magfura-2, respectively). The calcium release induced by thapsigargin (a Ca(2+)-ATPase inhibitor) was lower after puromycin treatment. Puromycin also reduced the [Ca(2+)](ER) level when perfused into the medium, but was ineffective after anisomycin pre-treatment (an inhibitor of the peptidyl transferase). Puromycin had a similar effect in the presence of heparin and ryanodine. This puromycin-evoked [Ca(2+)](ER) decrease was specific to the translocon. We conclude that the translocon complex is a major calcium leak channel. This work reveals a new role for the translocon which is involved in the control of the [Ca(2+)](ER) and could therefore supervise many physiological processes, including gene expression and apoptosis.

The mitochondrion is a privileged target for apopotosis-modulatory proteins of viral origin. Thus, viral protein R (Vpr) can target mitochondria and induce apoptosis via a specific interaction with the permeability transition pore complex (PTPC). Vpr cooperates with the adenine nucleotide translocator (ANT) to form large conductance channels and to trigger all the hallmarks of mitochondrial membrane permeabilization (MMP). The Vpr/ANT interaction is direct, since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT, ADP, ATP, or by Bcl-2. Accordingly, Vpr modulates MMP through direct structural and functional interactions with PTPC proteins.

Pleurotolysin, a sphingomyelin-specific cytolysin consisting of A (17 kDa) and B (59 kDa) components from the basidiomycete Pleurotus ostreatus, assembles into a transmembrane pore complex. Here, we cloned complementary and genomic DNAs encoding pleurotolysin, and studied pore-forming properties of recombinant proteins. The genomic regions encoding pleurotolysin A and B contained two and eight introns, respectively, and putative promoter sequences. The complementary DNA (cDNA) for pleurotolysin A encoded 138 amino acid residues, and the predicted product was identical with natural pleurotolysin A, except for the presence of the first methionine. Recombinant pleurotolysin A lacking the first methionine was purified as a 17-kDa protein with sphingomyelin-binding activity. The cDNA for pleurotolysin B encoded a precursor consisting of 523 amino acid residues, of which N-terminal 48 amino acid residues were absent in natural pleurotolysin B. Mature and precursor forms of pleurotolysin B were expressed as insoluble 59- and 63-kDa proteins, respectively, which were unfolded with 8 M urea and refolded by 100-fold dilution with 10 mM Tris–HCl buffer, pH 8.5. Although neither recombinant pleurotolysin A nor B alone was hemolytically active at higher concentrations of up to 100 mg/ml, they cooperatively assembled into a membrane pore complex on human erythrocytes and lysed the cell as efficiently as the natural proteins at nanomolar concentrations. In contrast, the precursor of pleurotolysin B was much less hemolytically active than mature pleurotolysin B in the presence of pleurotolysin A.

B16 melanoma (B16M) cells with high GSH content show high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (approximately 5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from approximately 19% (controls) to approximately 97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). l-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of l-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/Tet-Bcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to approximately 60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis.

The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain-linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membrane-permeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipid-linked disaccharide in the activity of nisin. We show here that Lipid II is not only the receptor for nisin but an intrinsic component of the pore formed by nisin, and we present a new model for the pore complex that includes Lipid II.

Plant mitochondria are remarkable with respect to their content in foreign, alien and plasmid-like DNA, raising the question of the transfer of this information into the organelles. We demonstrate the existence of an active, transmembrane potential-dependent mechanism of DNA uptake into plant mitochondria. The process is restricted to double-strand DNA, but has no obvious sequence specificity. It is most efficient with linear fragments up to a few kilobase pairs. When containing appropriate information, imported sequences are transcribed within the organelles. The uptake likely involves the voltage-dependent anion channel and the adenine nucleotide translocator, i.e. the core components of the mitochondrial permeability transition pore complex in animal cells, but it does not rely on known mitochondrial membrane permeabilization processes. We conclude that DNA import into plant mitochondria might represent a physiological phenomenon with some functional relevance.

The genomes of many viruses traffic into the nucleus, where they are either integrated into host chromosomes or maintained as episomal DNA and then transcriptionally activated or silenced. Here, we discuss the existing evidence on how the lentiviruses, adenoviruses, herpesviruses, hepadnaviruses and autonomous parvoviruses enter the nucleus. Depending on the size of the capsid enclosing the genome, three principles of viral nucleic acids import are discussed. The first principle is that the capsid disassembles in the cytosol or in a docked state at the nuclear pore complex and a subviral genomic complex is trafficked through the pore. Second, the genome is injected from a capsid that is docked to the pore complex, and third, import factors are recruited to cytosolic capsids to increase capsid affinity to the pore complex, mediate translocation and allow disassembly in the nucleoplasm.

Background: Mitochondrial dysfunction is the major cause of various types of cancer, leading to death worldwide. The present study investigated the in silico binding potential of natural flavonoids and essential oils with human cyclophilin D (CyPD) protein. CyPD protein is a major molecular marker for apoptosis and has been reported to be elevated in oral carcinoma. Methods: PubChem database was used to check the efficacy of different active phytoconstituents (kaempferol, quercetin, eugenol, oxyresveratrol, tanshinone 2a, catechin, epicatechin, cinnamaldehyde, and emodin). These compounds were used as ligands to check their potential as anticancer agents against the inner mitochondrial membrane protein, CyPD. Docking studies were performed with the help of Discovery Studio 2.5 and Autodock. Emodin was used as a reference inhibitor to compare the results. Results: The binding energy (B.E.) of the reference inhibitor (known/established drug) emodin was observed −28.9 kcal/mol while novel inhibitors (catechin, cinnamaldehyde, epicatechin, eugenol, kaempferol, oxyresveratrol, quercetin, and tanshinone 2a) exhibited a range from −51.51 to −5.89 kcal/mol. Quercetin, kaempferol, and epicatechin (B.E.: −51.51, −34.79, and −30.62 kcal/mol, respectively) showed strong affinity as compared to reference inhibitor (B.E.: −28.9 kcal/mol). Conclusion: Quercetin, kaempferol, and epicatechin can be used as lead inhibitors against targeting CyPD. Abbreviations used: CyPD: Cyclophilin D, BE: Binding Energy, PTPC: Permeability transition pore complex, mPTP: Mitochondrial permeability transition pore.