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.

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.

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 (Gb) 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.

Verotoxin Receptor-Based Pathology and Therapies.

Verotoxin, VT (aka Shiga toxin,Stx) is produced by enterohemorrhagic (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 (Gb), a glycosphingolipid (GSL) (aka CD77, pk blood group antigen) of the globoseries of neutral GSLs, initiating lipid raft-dependent plasma membrane Gb 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.

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.

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

The capacity of HIV-1 to develop resistance to current drugs calls for innovative strategies to control this infection. We aimed at developing novel inhibitors of HIV-1 replication by targeting viral RNA processing-a stage dependent on conserved host processes. We previously reported that digoxin is a potent inhibitor of this stage.

Glycosphingolipid storage in Fabry mice extends beyond globotriaosylceramide and is affected by ABCB1 depletion.

Aim: Fabry disease is caused by α-galactosidase A deficiency leading to accumulation of globotriaosylceramide (Gb) in tissues. Clinical manifestations do not appear to correlate with total Gb levels. Studies examining tissue distribution of specific acyl chain species of Gb and upstream glycosphingolipids are lacking.

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization.

Suppression of Adenovirus Replication by Cardiotonic Steroids.

Unlabelled: 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.

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.

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.

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.

CD4(+) T-cells are unable to express the HIV natural resistance factor globotriosylceramide.

Globotriaosylceramide (Gb(3)) is a cell surface-expressed natural resistance factor for HIV infection, but, its expression in human T-cells remains unknown. Therefore, Gb(3) in resting or activated CD4(+) T-cells was assessed by flow cytometry and thin layer chromatography of cell extracts. We found the majority of CD4(+) T-cells, whether resting or activated, do not express Gb(3) at significant levels (<2% positive cells).

Verotoxin A subunit protects lymphocytes and T cell lines against X4 HIV infection in vitro.

Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb(3) (globotriaosylceramide, Pk blood group antigen) as a natural resistance factor for HIV infection. Gb(3) is a B cell marker (CD77), but a fraction of activated peripheral blood mononuclear cells (PBMCs) can also express Gb(3). Activated PBMCs predominantly comprise CD4+ T-cells, the primary HIV infection target.

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.

Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane.

The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin-Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes.

Comparison of detection methods for cell surface globotriaosylceramide.

The cell surface-expressed glycosphingolipid (GSL), globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding.

Glycosphingolipid functions.

The combination of carbohydrate and lipid generates unusual molecules in which the two distinctive halves of the glycoconjugate influence the function of each other. Membrane glycolipids can act as primary receptors for carbohydrate binding proteins to mediate transmembrane signaling despite restriction to the outer bilayer leaflet. The extensive heterogeneity of the lipid moiety plays a significant, but still largely unknown, role in glycosphingolipid function.

The role of glycosphingolipids in HIV/AIDS.

Much remains unknown about basic aspects of HIV-1 infection and cell susceptibility. Glycosphingolipid (GSL) binding by the HIV-1 adhesin gp120 has long been implicated in the infection of non-lymphoid cells, as well as CD4(+) T cells and monocytes, the primary targets of HIV-1 infection. We have identified the P(k) blood group antigen (a GSL) globotriaosylceramide (Gb(3)) as a new resistance effector against HIV-1 infection.

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.

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.

Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells.

Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization.

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.

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.

New aspects of the regulation of glycosphingolipid receptor function.

We propose that the fatty acid heterogeneity of glycosphingolipids may compensate for the relative few and simple glycosphingolipid structures found in mammalian cells. Variation in GSL fatty acid composition may mediate aglycone regulation of GSL membrane receptor function by a differential interaction with cholesterol and other membrane components which may be differentially organized within plasma membrane lipid domains. These concepts are specifically illustrated in model membrane studies and in relation to the role of the glycolipid, globotriaosyl ceramide (Gb(3)) in verotoxin-induced renal pathology and gp120 binding in HIV infection.