A cyclodextrin dimer with a photocleavable linker as a possible carrier for the photosensitizer in photodynamic tumor therapy

A cyclodextrin dimer has been synthesized with two β-cyclodextrins linked by a flexible chain containing a carbon–carbon double bond. This dimer binds and solubilizes a phthalocyanine-based photosensitizer that generates singlet oxygen on irradiation. When the complex is irradiated, the singlet oxygen cleaves the carbon–carbon link, and the cyclodextrins are released, liberating the photosensitizer into the light path. Ideas about how this phenomenon could be used to make photodynamic tumor therapy into a more selective process are described.

Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

Selective disruption of protein aggregation by cyclodextrin dimers

β-Cyclodextrin (CD) dimers (n = 11) were synthesized and tested against eight enzymes, seven of which were dimeric or tetrameric, for inhibitor activity. Initial screening showed that only l-lactate dehydrogenase and citrate synthase were inhibited but only by two specific CD dimers in which two β-CDs were linked on the secondary face by a pyridine-2,6-dicarboxylic group. Further investigation suggested that these CD dimers inhibit the activity of l-lactate dehydrogenase and citrate synthase at least in part by disruption of protein–protein aggregation.

An artificial cytochrome P450 that hydroxylates unactivated carbons with regio- and stereoselectivity and useful catalytic turnovers

A catalyst has been synthesized comprising a manganese porphyrin carrying four beta-cyclodextrin groups. It catalyzes the hydroxylation of substrates of appropriate size carrying tert-butylphenyl groups that can hydrophobically bind into the cyclodextrin cavities. In one example as many as 650 catalytic turnovers are seen before the catalyst is oxidatively destroyed, and with a rate comparable to that of typical cytochrome P450 enzymes. In another example, a steroid derivative is regio- and stereoselectively hydroxylated at a single unactivated carbon atom, but more slowly and with fewer turnovers. The carbon attacked is not the most chemically reactive, and the selectivity is determined by the geometry of the catalyst-substrate complex. Nonbinding substrates are not reactive under the conditions used, and substrates with more flexible binding geometries give more than a single product.

Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15°C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.

Cholesterol depletion inhibits the generation of β-amyloid in hippocampal neurons

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer’s disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called β-amyloid (Aβ) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Aβ that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-β-cyclodextrin, we show that the formation of Aβ is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Aβ formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Aβ formation to occur and imply a link between cholesterol, Aβ, and Alzheimer’s disease.

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCθ) of perfringolysin O (θ-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCθ on a sucrose gradient. BCθ was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCθ binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCθ binding to FLDF. The staining patterns of BCθ and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCθ binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCθ binding does not cause any damage to cell membranes, indicating that BCθ is a useful probe for the detection of membrane rafts in living cells.

Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the α-secretase ADAM 10

Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer's disease. In the present report, we identify the α-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neural cell lines with either the cholesterol-extracting agent methyl-β-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted α-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of α-secretase activity resulted in a decreased secretion of Aβ peptides. Several mechanisms were elucidated as being the basis of enhanced α-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-β-cyclodextrin; treatment with lovastatin resulted in higher expression of the α-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic α-secretase pathway and the formation of neuroprotective α-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer's disease.

Sphingomyelin-enriched Microdomains at the Golgi Complex

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the α- and β-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.