Delta-toxin from Clostridium perfringens perturbs intestinal epithelial barrier function in Caco-2 cell monolayers

Clostridium perfringens delta-toxin is a β-barrel-pore-forming toxin (β-PFT) and a presumptive virulence factor of type B and C strains, which are causative organisms of fatal intestinal diseases in animals. We showed previously that delta-toxin causes cytotoxicity via necrosis in sensitive cells. Here, we examined the effect of delta-toxin on intestinal membrane integrity. Delta-toxin led to a reduction in transepithelial electrical resistance (TEER) and increased the permeability of fluorescence isothiocyanate-conjugated dextran in human intestinal epithelial Caco-2 cells without changing the tight junction proteins, such as zonula occludens-1 (ZO-1), occludin, and claudin-1. On the other hand, delta-toxin reduced the cellular levels of adherence junction protein E-cadherin before cell injury. A disintegrin and metalloprotease (ADAM) 10 facilitates E-cadherin cleavage and was identified as the cellular receptor for alpha-toxin, a β-PFT produced by Staphylococcus aureus. ADAM10 inhibitor (GI254023X) blocked the toxin-induced decrease in TEER and cleavage of E-cadherin. Delta-toxin enhanced ADAM10 activity in a dose- and time-dependent manner. Furthermore, delta-toxin colocalized with ADAM10. These results indicated that ADAM10 plays a key role in delta-toxin-induced intestinal injury.     

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present G_M2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.     

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.     

Lipid raft disruption protects mature neurons against amyloid oligomer toxicity

A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid β oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca²⁺ rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.     

Oligomerization of Clostridium perfringens Epsilon Toxin Is Dependent upon Caveolins 1 and 2

Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization – an event which is requisite for pore formation and, by extension, cell death.     

Prion Protein-mediated Toxicity of Amyloid-β Oligomers Requires Lipid Rafts and the Transmembrane LRP1

Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrP^C) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrP^C. The binding of Aβ oligomers to cell surface PrP^C, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrP^C, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrP^C impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrP^C-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrP^C in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD.     

Macrophage cytotoxicity produced by adenylate cyclase toxin from Bordetella pertussis: more than just making cyclic AMP!

The cytotoxic effect of adenylate cyclase (AC) toxin from Bordetella pertussis on host cells has been attributed to the production of supraphysiologic levels of cyclic AMP by the toxin. We have tested this hypothesis and show that at least two different mechanisms, cAMP accumulation/ATP depletion and oligomerization/pore formation, contribute, perhaps synergistically, to AC toxin-induced cytotoxicity. Wild-type (WT) AC toxin causes cell death associated with an increase in cAMP, a reduction in ATP, activation of caspases 3/7, and increased annexin V and TUNEL staining. In contrast, a non-acylated, enzymatically active, non-haemolytic form of AC toxin is able to increase cAMP, reduce ATP and elicit annexin V staining, but the decrease in ATP and the annexin staining are transient and there is minimal caspase activation, TUNEL staining and cell death. Mutant AC toxins defective in either enzymatic activity or the ability to deliver their enzymatic domain are able to kill J774 cells, without cAMP production, and with minimal caspase activation and TUNEL staining. Comparison of the potencies of WT toxin and those of mutants that only increase cAMP or only create transmembrane pores establishes that at least two mechanisms are contributory and that simply the production of cAMP is not enough to account for the cytotoxicity produced by AC toxin.     

Hemolysis induced by Bacillus cereus sphingomyelinase

Bacillus cereus sphingomyelinase (Bc-SMase) induces hemolysis of sheep erythrocytes which contain large amounts of sphingomyelin. We investigated the mechanism of this hemolysis in comparison to that induced by Clostridium perfringens alpha-toxin. Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a ceramidase inhibitor, and dihydrosphingosine, a sphingosine kinase inhibitor, did not inhibit the hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the detergent-resistant membrane fractions, lipid rafts. The level of ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the hemolysis by Bc-SMase was the same as that by the toxin. However, the level of ceramide production induced by Bc-SMase in SM-liposomes was equal to that triggered by the toxin, when the carboxyl fluorescein-release from liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced hemolysis of sheep erythrocytes is related to the formation of interface between ceramide-rich domains and ceramide-poor domains through production of ceramide from SM.     

Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-β-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na⁺ and N-methyl-d-glucamine (NMDG⁺) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.     

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.     

Clostridium perfringens Delta Toxin Is Sequence Related to Beta Toxin,NetB, and Staphylococcus Pore-Forming Toxins,but Shows Functional Differences

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside G_M2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to G_M2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes G_M2 as receptor and forms anion-selective channels.     

Toxigenic Profile of Clostridium perfringens Strains Isolated from Natural Ingredient Laboratory Animal Diets

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium that ubiquitously inhabits a wide variety of natural environments including the gastrointestinal tract of humans and animals. C. perfringens is an opportunistic enteropathogen capable of producing at least 20 different toxins in various combinations. Strains of C. perfringens are currently categorized into 7 toxinotypes (A, B, C, D, E, F, and G) based on the presence or absence of 6 typing-toxins (α, β, epsilon, iota, enterotoxin, and netB). Each toxinotype is associated with specific histotoxic and enteric diseases. Spontaneous enteritis due to C. perfringens has been reported in laboratory animals; however, the source of the bacteria was unknown. The Quality Assurance Laboratory (QAL) at the National Institute of Environmental Health Sciences (NIEHS) routinely screens incoming animal feeds for aerobic, enteric pathogens, such as Salmonella spp. and E. coli. Recently, QAL incorporated anaerobic screening of incoming animal feeds. To date, the lab has isolated numerous Clostridium species, including C. perfringens, from 23 lots of natural ingredient laboratory animal diets. Published reports of C. perfringens isolation from laboratory animal feeds could not be found in the literature. Therefore, we performed a toxin profile screen of our isolated strains of C. perfringens using PCR to determine which toxinotypes were present in the laboratory animal diets. Our results showed that most C. perfringens strains we isolated from the laboratory animal feed were toxinotype A with most strains also possessing the theta toxin. Two of the C. perfringens strains also possessed the β toxin. Our results demonstrated the presence of C. perfringens in nonsterile, natural ingredient feeds for laboratory animals which could serve as a source of this opportunistic pathogen.     

Calcium enhances binding of Clostridium perfringens epsilon toxin to sulfatide

Epsilon toxin (Etx) from Clostridium perfringens is synthesized as a very low-active prototoxin form (proEtx) that becomes active upon proteolytic activation and has the capacity to cross the blood-brain barrier (BBB), thereby producing severe neurological effects. The identity and requirements of host receptors of Etx remain a matter of controversy. In the present study, we analysed the binding of proEtx or Etx to liposomes containing distearoylphosphatidylcholine (DSPC), cholesterol and sulfatide, or alternatively to detergent-solubilized lipids, using surface plasmon resonance (SPR). We also tested the influence of calcium on Etx or proEtx binding. Our findings show that the presence of sulfatide in liposomes increases both Etx and proEtx binding, and Etx binding is enhanced by calcium. These results were corroborated when SPR was conducted with immobilized toxin, since detergent-solubilized sulfatide increases its binding to Etx in the presence of calcium, but not to proEtx. Moreover, binding affinity is also affected, since the treatment of liposomes with sulfatase causes the dissociation rate constants (K_D) in both proEtx and Etx to increase, especially in the case of proEtx in the presence of calcium. In addition, protein-lipid overlay assays corroborated the calcium-induced enhancement of Etx binding to sulfatide, and to lipids extracted from sulfatide-enriched rat brain lipid rafts. In conclusion, the present work highlights the role of sulfatide as an important element in the pathophysiology of Etx and reveals the influence of calcium in the interaction of Etx, but not of proEtx, with the target membrane.     

用鞘磷脂作为标志分子鉴定脂筏

脂筏是质膜双层中富含鞘脂、胆固醇及特殊蛋白质的质膜微区.对其功能的研究,首先要对其进行分离和鉴定.常利用密度梯度超速离心将其分离,然后以脂筏中富含的神经节苷脂GM₁作为标志分子,利用荧光或生物素标记的霍乱毒素-B亚基进行亲和标记来鉴定脂筏.但这一鉴定方法操作复杂、费时、易对环境造成污染,所用关键试剂霍乱毒素不易获得,再加上一些组织GM₁含量甚微或不含GM₁,使其应用受到局限.为建立一个特异性高又对各种组织广泛适应的脂筏鉴定方法.对两种细胞系脂筏的脂类组分进行了分析.结果发现,可用鞘磷脂作为脂筏的特异性标志分子,采用高效薄层层析技术对脂筏进行鉴定.     

Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin.

When cell lines that are susceptible to diphtheria toxin, such as human FL cells, were treated with C. perfringens neuraminidase their sensitivities to the toxin were increased. The sensitivities of the cells to the toxin were also increased by treatment with neuraminidase from Arthrobacter ureafaciens or HVJ (Sendai virus). Neuraminidase did not have this effect on a toxin-resistant cell line. It also did not increase the cytotoxic effect of a large concentration of fragment A of diphtheria toxin, which lacks the moiety of the toxin molecule that binds to the cell membrane. Neuraminidase from C. perfringens or HVJ also increased the sensitivity of cells to ricin toxin. Furthermore, neuraminidase from C. perfringens or A. ureafaciens increased the sensitivity of cells to Pseudomonas aeruginosa exotoxin (PA toxin), but in this case neuraminidase from HVJ did not have a similar effect.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44⁺ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.     

Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22a_Q64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22a_Q64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.     

Raft-targeting and oligomerization of Parasporin-2, a bacillus thuringiensis crystal protein with anti-tumour activity

Parasporin-2 is a newly classified Bacillus thuringiensis crystal toxin with strong cytocidal activities toward human liver and colon cancer cells. Similar to other insecticidal B. thuringiensis crystal toxins, parasporin-2 shows target specificity and damages the cellular membrane. However, the mode of parasporin-2 actions toward the cell membrane remains unknown. Here, we show that this anti-tumour crystal toxin targets lipid rafts and assembles into oligomeric complexes in the membrane of human hepatocyte cancer (HepG2) cells. Upon incubation with HepG2 cells, peripheral membrane-bound toxins, which were recovered in a low-density detergent-resistant membrane fraction, i.e. with lipid rafts, were transformed into heat-stable SDS-resistant membrane-embedded oligomers (approximately 200 kDa). The toxin oligomerization was dependent on temperature and coupled with cell lysis. The toxin oligomerization also occurred in a cell-free membrane system and was required for binding to membrane proteins, the lipid bilayer and cholesterols. These results indicate that parasporin-2 is an oligomerizing and pore-forming toxin that accumulates in lipid rafts.     

GRIN1 Regulates ��-Opioid Receptor Activities by Tethering the Receptor and G Protein in the Lipid Raft

The lipid raft location of μ-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein α-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence ²⁶⁷GSKEK²⁷¹ within the MOR third intracellular loop that is not involved in Gα interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Gα interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Gα. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-β-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Gα with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.