Is cholesterol both the lock and key to abnormal transmembrane signals in Autism Spectrum Disorder?

Disturbances in cholesterol homeostasis have been associated with ASD. Lipid rafts are central in many transmembrane signaling pathways (including mTOR) and changes in raft cholesterol content affect their order function. Cholesterol levels are controlled by several mechanisms, including endoplasmic reticulum associated degradation (ERAD) of the rate limiting HMGCoA reductase. A new approach to increase cholesterol via temporary ERAD blockade using a benign bacterial toxin-derived competitor for the ERAD translocon is suggested.A new lock and key model for cholesterol/lipid raft dependent signaling is proposed in which the rafts provide both the afferent and efferent 'tumblers' across the membrane to allow 'lock and key' receptor transmembrane signals.

ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species.

The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.

Suppression of Adenovirus Replication by Cardiotonic Steroids.

The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies. IMPORTANCE: Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

Synthesis of a novel photoactivatable glucosylceramide cross-linker.

The biosynthesis of glucosylceramide (GlcCer) is a key rate-limiting step in complex glycosphingolipid (GSL) biosynthesis. To further define interacting partners of GlcCer, we have made a cleavable, biotinylated, photoreactive GlcCer analog in which the reactive nitrene is closely apposed to the GlcCer head group, by substituting the native fatty acid with d, l-2-aminohexadecanoic acid. Two amino-GlcCer diastereomer cross-linkers (XLA and XLB) were generated. XLB proved an effective lactosylceramide (LacCer) synthase substrate while XLA was inhibitory. Both probes specifically bound and cross-linked the GlcCer binding protein, glycolipid transfer protein (GLTP), but not other GSL binding proteins (Shiga toxin and cholera toxin). GlcCer inhibited GLTP cross-linking. Both GlcCer cross-linkers competed with microsomal nitrobenzoxadiazole (NBD)-GlcCer anabolism to NBD-LacCer. GLTP showed marked, ATP-dependent enhancement of cell-free intact microsomal LacCer synthesis from endogenous or exogenous liposomal GlcCer, supporting a role in the transport/membrane translocation of cytosolic and extra-Golgi GlcCer. GLTP was specifically labeled by either XLA or XLB GlcCer cross-linker during this process, together with a (the same) small subset of microsomal proteins. These cross-linkers will serve to probe physiologically relevant GlcCer-interacting cellular proteins.

Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling.

Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcß1-3Galß1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism.

Structure-dependent pseudoreceptor intracellular traffic of adamantyl globotriaosyl ceramide mimics.

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.

Cholesterol masks membrane glycosphingolipid tumor-associated antigens to reduce their immunodetection in human cancer biopsies.

Glycosphingolipids (GSLs) are neoplastic and normal/cancer stem cell markers and GSL/cholesterol-containing membrane rafts are increased in cancer cell plasma membranes. We define a novel means by which cancer cells can restrict tumor-associated GSL immunoreactivity. The GSL-cholesterol complex reorients GSL carbohydrate to a membrane parallel, rather than perpendicular conformation, largely unavailable for antibody recognition. Methyl-ß-cyclodextrin cholesterol extraction of all primary human tumor frozen sections tested (ovarian, testicular, neuroblastoma, prostate, breast, colon, pheochromocytoma and ganglioneuroma), unmasked previously "invisible" membrane GSLs for immunodetection. In ovarian carcinoma, globotriaosyl ceramide (Gb3), the GSL receptor for the antineoplastic Escherichia coli-derived verotoxin, was increased throughout the tumor. In colon carcinoma, Gb3 detection was vastly increased within the neovasculature and perivascular stroma. In tumors considered Gb3 negative (neuroblastoma, Leydig testicular tumor and pheochromocytoma), neovascular Gb3 was unmasked. Tumor-associated GSL stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4 and globoH were unmasked according to tumor: SSEA-1 in prostate/colon; SSEA-3 in prostate; SSEA-4 in pheochromocytoma/some colon tumors; globoH in prostate/some colon tumors. In colon, anti-SSEA-1 was tumor cell specific. Within the GSL-cholesterol complex, filipin-cholesterol binding was also reduced. These results may relate to the ill-defined benefit of statins on cancer prognosis, for example, prostate carcinoma. We found novel anti-tumor GSL antibodies circulating in 3/5 statin-treated, but not untreated, prostate cancer patients. Lowering tumor membrane cholesterol may permit immune recognition of otherwise unavailable tumor-associated GSL carbohydrate, for more effective immunosurveillance and active/passive immunotherapy. Our results show standard immunodetection of tumor GSLs significantly under assesses tumor membrane GSL content, impinging on the current use of such antigens as cancer vaccines.

Cholesterol modulates glycolipid conformation and receptor activity.

We document a new dimension of surface recognition in which communication is controlled through the collective behavior of lipids. Membrane cholesterol induces a tilt in glycolipid receptor headgroup, resulting in loss of access for ligand binding. This property appears to organize erythrocyte blood group presentation and glycolipid receptor function during the activation of sperm fertility, suggesting that lipid 'allostery' is a means to regulate membrane recognition processes.

Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism.

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 µM adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 µM adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 µM adaGalCer, cell synthesis of only Gb(3) and Gb(4) was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb(2)), was generated, indicating substrate competition for Gb(3) synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb(3) synthesis was inhibited by adaGalCer. Metabolic labeling of Gb(3) in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb(2) was produced. AdaGb(2) in cells was 10-fold more effectively shed into the medium than the more polar Gb(3), providing an easily eliminated "safety valve" alternative to Gb(3) accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.

A major fraction of glycosphingolipids in model and cellular cholesterol-containing membranes is undetectable by their binding proteins.

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based "aglycone" interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb(3)), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb(3/)cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb(3), HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb(3), more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb(3)/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb(3), suggesting GSL-GSL interaction can counter cholesterol masking of Gb(3). The similar separation of Vero cell membrane-derived vesicles into minor "binding," and major "non-binding" fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of "available" and "cholesterol-masked" plasma membrane Gb(3) pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.

The human P(k) histo-blood group antigen provides protection against HIV-1 infection.

Several human histo-blood groups are glycosphingolipids, including P/P1/P(k). Glycosphingolipids are implicated in HIV-host-cell-fusion and some bind to HIV-gp120 in vitro. Based on our previous studies on Fabry disease, where P(k) accumulates and reduces infection, and a soluble P(k) analog that inhibits infection, we investigated cell surface-expressed P(k) in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P(1)(k), where P(k) is overexpressed, or blood group p, that completely lacks P(k), were compared with draw date-matched controls. Fluorescence-activated cell sorter analysis and/or thin layer chromatography were used to verify P(k) levels. P(1)(k) PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10- to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P(k), but not CD4 or chemokine coreceptor expression, correlated with infection. P(k) liposome-fused cells and CD4(+) HeLa cells manipulated to express high or low P(k) levels confirmed a protective effect of P(k). We conclude that P(k) expression strongly influences susceptibility to HIV-1 infection, which implicates P(k) as a new endogenous cell-surface factor that may provide protection against HIV-1 infection.

Therapeutic Uses of Bacterial Subunit Toxins.

The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb3) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin-receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb3 (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb3 is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb3 is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing E. coli infection. The Gb3 levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb3 is upregulated in the neovasculature of many tumours, irrespective of tumour Gb3 status. Gb3 is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented.

A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms.

Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb(3)/P(k)/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb(3)/P(k) (FSL-Gb(3)) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb(3), of the natural Gb(3)/P(k) molecule was synthesized. HIV-1(IIIB) (X4 virus) and HIV-1(Ba-L) (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb(3). We monitored Gb(3), CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24(gag) ELISA. Total cellular Gb(3) was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb(3) blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC(50)) of approximately 200-250 microM. FACS and TLC overlay showed that FSL-Gb(3) can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb(3) is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb(3) on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb(3) can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb(3) into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb(3), having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS.

Verotoxin Receptor-Based Pathology and Therapies.

Verotoxin, VT (aka Shiga toxin,Stx) is produced by enterohemorrhagic E. coli (EHEC) and is the key pathogenic factor in EHEC-induced hemolytic uremic syndrome (eHUS-hemolytic anemia/thrombocytopenia/glomerular infarct) which can follow gastrointestinal EHEC infection, particularly in children. This AB5 subunit toxin family bind target cell globotriaosyl ceramide (Gb3), a glycosphingolipid (GSL) (aka CD77, pk blood group antigen) of the globoseries of neutral GSLs, initiating lipid raft-dependent plasma membrane Gb3 clustering, membrane curvature, invagination, scission, endosomal trafficking, and retrograde traffic via the TGN to the Golgi, and ER. In the ER, A/B subunits separate and the A subunit hijacks the ER reverse translocon (dislocon-used to eliminate misfolded proteins-ER associated degradation-ERAD) for cytosolic access. This property has been used to devise toxoid-based therapy to temporarily block ERAD and rescue the mutant phenotype of several genetic protein misfolding diseases. The A subunit avoids cytosolic proteosomal degradation, to block protein synthesis via its RNA glycanase activity. In humans, Gb3 is primarily expressed in the kidney, particularly in the glomerular endothelial cells. Here, Gb3 is in lipid rafts (more ordered membrane domains which accumulate GSLs/cholesterol) whereas renal tubular Gb3 is in the non-raft membrane fraction, explaining the basic pathology of eHUS (glomerular endothelial infarct). Females are more susceptible and this correlates with higher renal Gb3 expression. HUS can be associated with encephalopathy, more commonly following verotoxin 2 exposure. Gb3 is expressed in the microvasculature of the brain. All members of the VT family bind Gb3, but with varying affinity. VT2e (pig edema toxin) binds Gb4 preferentially. Verotoxin-specific therapeutics based on chemical analogs of Gb3, though effective in vitro, have failed in vivo. While some analogs are effective in animal models, there are no good rodent models of eHUS since Gb3 is not expressed in rodent glomeruli. However, the mouse mimics the neurological symptoms more closely and provides an excellent tool to assess therapeutics. In addition to direct cytotoxicity, other factors including VT-induced cytokine release and aberrant complement cascade, are now appreciated as important in eHUS. Based on atypical HUS therapy, treatment of eHUS patients with anticomplement antibodies has proven effective in some cases. A recent switch using stem cells to try to reverse, rather than prevent VT induced pathology may prove a more effective methodology.

Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes.

Glycosphingolipid (GSL) fatty acid strictly regulates verotoxin 1 (VT1) and the HIV adhesin, gp120 binding to globotriaosyl ceramide within Gb(3)/cholesterol detergent resistant membrane (DRM) vesicle constructs and in Gb(3) water-air interface monolayers in a similar manner. VT2 bound Gb(3)/cholesterol vesicles irrespective of fatty acid composition, but VT1 bound neither C18 nor C20Gb(3)vesicles. C18/C20Gb(3) were dominant negative in mixed Gb(3) fatty acid isoform vesicles, but including C24:1Gb(3) gave maximal binding. VT1 bound C18Gb(3) vesicles after cholesterol removal, but C20Gb(3)vesicles required sphingomyelin in addition for binding. HIV-1gp120 also bound C16, C22, and C24, but neither C18 nor C20Gb(3) vesicles. C18 and C20Gb(3) were, in mixtures without C24:1Gb(3), dominant negative for gp120 vesicle binding. Gp120/VT1bound C18 and C24:1Gb(3) mixtures, although neither isoform bound alone. Monolayer surface pressure measurement showed VT1, but not VT2, bound Gb(3) at cellular DRM surface pressures, and confirmed loss of VT1 and gp120 (but not VT2) specific C18Gb(3) binding. We conclude fatty-acid mediated fluidity within simple model GSL/cholesterol DRM can selectively regulate GSL carbohydrate-ligand binding.

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels.

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide (Gb(3), CD77, p(k) blood group antigen) in HIV-1 infection, we have pharmacologically modulated Gb(3) metabolism in an X4 HIV-1 infectable monocytic cell line (THP-1) that naturally expresses Gb(3) and in a Gb(3)-expressing glioblastoma cell line (U87) transfected to express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1 and U87 cells were treated with either a competitive inhibitor of alpha-galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induce Gb(3) accumulation, or a glucosylceramide synthase inhibitor, phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to deplete cells of Gb(3). HIV susceptibility was determined via measurement of p24(gag) antigen production by ELISA. In addition, total cellular Gb(3) content was determined using thin layer chromatography followed by Verotoxin1 overlay binding. The cell surface expression of Gb(3) was verified by FACS analysis. We found that DGJ significantly decreased THP-1 and U87 cell susceptibility to HIV-1(IIIB) and HIV-1(BaL) infection, respectively, at a concentration of approximately 100 microM. In contrast, P4 (2 microM) substantially increased cellular susceptibility to HIV-1 infection. Total cellular GSL analysis verified increased Gb(3) expression in cells treated with DGJ and considerable reduction of Gb(3) in P4-treated cells as compared to controls. These results show a reciprocal relationship between Gb(3) expression and infection with either X4 HIV-1(IIIB) or R5 HIV-1(Ba-L). These results support previous studies that Gb(3) provides resistance to HIV infection. Variable Gb(3) expression may provide a natural HIV resistance factor in the general population, and pharmacological manipulation of Gb(3) levels may provide an approach to induction of HIV resistance.

Detergent-resistant globotriaosyl ceramide may define verotoxin/glomeruli-restricted hemolytic uremic syndrome pathology.

Verotoxin binding to its receptor, globotriaosyl ceramide(Gb(3)) mediates the glomerular pathology of hemolytic uremic syndrome, but Gb(3) is expressed in both tubular and glomerular cells. Gb(3) within detergent-resistant membranes, an index of glycolipid-cholesterol enriched lipid rafts, is required for in vitro cytotoxicity. We found that verotoxin 1 and 2 binding to human adult renal glomeruli is detergent resistant, whereas the strong verotoxin binding to renal tubules is detergent sensitive. Verotoxin binding to pediatric glomeruli was detergent resistant but binding to adult glomeruli was enhanced, remarkably for some samples, by detergent extraction. Detergent-sensitive glomerular components may provide age-related protection against verotoxin glomerular binding. Mouse glomeruli remained verotoxin unreactive after detergent extraction, whereas tubular binding was lost. Cholesterol extraction induced strong verotoxin binding in poorly reactive adult glomeruli, suggesting cholesterol can mask Gb(3) in glomerular lipid rafts. Binding of the human immunodeficiency virus (HIV) adhesin, gp120 (another Gb(3) ligand) was detergent sensitive, tubule-restricted, and inhibited by verotoxin B subunit pretreatment, and may relate to HIV nephropathy. Our study shows that differential membrane Gb(3) organization in glomeruli and tubules provides a basis for the age- and glomerular-restricted pathology of hemolytic uremic syndrome.

Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen.

Polyomaviruses such as BK virus and JC virus have been linked to several diseases, but treatments that thwart their propagation are limited in part because of slow growth and cumbersome culturing conditions. In contrast, the replication of one member of this family, Simian Virus 40 (SV40), is robust and has been well-characterized. SV40 replication requires two domains within the viral-encoded large tumor antigen (TAg): The ATPase domain and the N-terminal J domain, which stimulates the ATPase activity of the Hsp70 chaperone. To assess whether inhibitors of polyomavirus replication could be identified, we examined a recently described library of small molecules, some of which inhibit chaperone function. One compound, MAL2-11B, inhibited both TAg's endogenous ATPase activity and the TAg-mediated activation of Hsp70. MAL2-11B also reduced SV40 propagation in plaque assays and compromised DNA replication in cell culture and in vitro. Furthermore, the compound significantly reduced the growth of BK virus in a human kidney cell line. These data indicate that pharmacological inhibition of TAg's chaperone and ATPase activities may provide a route to combat polyomavirus-mediated disease.

Author Correction: Cardiac glycoside/aglycones inhibit HIV-1 gene expression by a mechanism requiring MEK1/2-ERK1/2 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Shiga toxin 2 causes apoptosis in human brain microvascular endothelial cells via C/EBP homologous protein.

Shiga toxin 1 (Stx1) and Stx2 produced by Escherichia coli O157 are known to be cytotoxic to Vero and HeLa cells by inhibiting protein synthesis and by inducing apoptosis. In the present study, we have demonstrated that 10 ng/ml Stx2 induced DNA fragmentation in human brain microvascular endothelial cells (HBMEC), with cleavage activation of caspase-3, -6, -8, and -9. A microarray approach used to search for apoptotic potential signals in response to Stx2 revealed that Stx2 treatment induced a marked upregulation of C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible protein 153 (GADD153). Increased CHOP expression was dependent on enzymatically active Stx1. Knockdown of CHOP mRNA reduced the activation of caspase-3 and prevented apoptotic cell death. These results suggest that Stx2-induced apoptosis is mediated by CHOP in HBMEC and involves activation of both the intrinsic and extrinsic pathways of apoptosis.