Yvonne Lange

Probing red cell membrane cholesterol movement with cyclodextrin.

We probed the kinetics with which cholesterol moves across the human red cell bilayer and exits the membrane using methyl-beta-cyclodextrin as an acceptor. The fractional rate of cholesterol transfer (% s(-1)) was unprecedented, the half-time at 37 degrees C being ~1 s. The kinetics observed under typical conditions were independent of donor concentration and directly proportional to acceptor concentration. The rate of exit of membrane cholesterol fell hyperbolically to zero with increasing dilution. The energy of activation for cholesterol transfer was the same at high and low dilution; namely, 27-28 Kcal/mol. This behavior is not consistent with an exit pathway involving desorption followed by aqueous diffusion to acceptors nor with a simple one-step collision mechanism. Rather, it is that predicted for an activation-collision mechanism in which the reversible partial projection of cholesterol molecules out of the bilayer precedes their collisional capture by cyclodextrin. Because the entire membrane pool was transferred in a single first-order process under all conditions, we infer that the transbilayer diffusion (flip-flop) of cholesterol must have proceeded faster than its exit, i.e., with a half-time of <1 s at 37 degrees C.

Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles.

Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP-C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile-treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g.trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7-ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease.

CIRCULATION OF CHOLESTEROL BETWEEN LYSOSOMES AND THE PLASMA MEMBRANE

The cholesterol in the lysosomes of cultured human fibroblasts was determined to constitute ∼6% of the cell total. This pool was enlarged by as much as 10-fold in Niemann-Pick type C cells. Certain amphiphiles (e.g. U18666A, progesterone, and imipramine) caused lysosomal cholesterol to increase to similarly high levels at a rate of ∼0.8% of cell cholesterol/h. Lysosomal cholesterol accumulated even in the absence of exogenous lipoproteins. Furthermore, nearly all of the lysosomal cholesterol in both of the two perturbed systems was shown to be derived from the plasma membrane. Oxysterols known to alter cholesterol movement and homeostasis blocked lysosomal cholesterol accretion in amphiphile-treated cells, suggesting that this process is regulated physiologically. Treating cells with amphiphiles slightly reduced the efflux of cholesterol from lysosomes and slightly increased the influx from the plasma membrane, causing the lysosomal cholesterol compartment to double in size in ∼15 h. After more prolonged amphiphile treatments, a population of buoyant lysosomes appeared that exchanged cholesterol with the plasma membrane completely but slowly. Niemann-Pick type C lysosomes were similarly buoyant and sluggish. We conclude that cholesterol circulates bidirectionally between the plasma membrane and lysosomes. The massive accumulation of lysosomal cholesterol in the perturbed cells does not appear to reflect disabled lysosomal transport but rather the formation of lysosomes modified for lipid storage, i.e. lamellar bodies.

CHOLESTEROL HOMEOSTASIS AND THE ESCAPE TENDENCY (ACTIVITY) OF PLASMA MEMBRANE CHOLESTEROL

We review evidence that sterols can form stoichiometric complexes with certain bilayer phospholipids, and sphingomyelin in particular. These complexes appear to be the basis for the formation of condensed and ordered liquid phases, (micro)domains and/or rafts in both artificial and biological membranes. The sterol content of a membrane can exceed the complexing capacity of its phospholipids. The excess, uncomplexed membrane sterol molecules have a relatively high escape tendency, also referred to as fugacity or chemical activity (and, here, simply activity). Cholesterol is also activated when certain membrane intercalating amphipaths displace it from the phospholipid complexes. Active cholesterol projects from the bilayer and is therefore highly susceptible to attack by cholesterol oxidase. Similarly, active cholesterol rapidly exits the plasma membrane to extracellular acceptors such as cyclodextrin and high-density lipoproteins. For the same reason, the pool of cholesterol in the ER (endoplasmic reticulum) increases sharply when cell surface cholesterol is incremented above the physiological set-point; i.e., equivalence with the complexing phospholipids. As a result, the escape tendency of the excess cholesterol not only returns the plasma membrane bilayer to its set-point but also serves as a feedback signal to intracellular homeostatic elements to down-regulate cholesterol accretion.

Quantitation of the Pool of Cholesterol Associated with Acyl-CoA:Cholesterol Acyltransferase in Human Fibroblasts

The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER)in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of [14C]oleoyl-CoA converted ∼0.1–2% of total cell-free cholesterol to [14C]cholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction. The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore appears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways.

Effectors of Rapid Homeostatic Responses of Endoplasmic Reticulum Cholesterol and 3-Hydroxy-3-methylglutaryl-CoA Reductase

The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol.

Salmonella enterica Serovar Typhimurium Requires Nonsterol Precursors of the Cholesterol Biosynthetic Pathway for Intracellular Proliferation

ABSTRACT We have previously shown that Salmonella enterica serovar Typhimurium infection perturbs the host cholesterol biosynthetic pathway. Here we show that inhibiting the first step of this pathway (3-hydroxy-3-methylglutaryl coenzyme A reductase) reduces the growth of intracellular S. enterica serovar Typhimurium and has no effect on extracellular bacterial growth. Selectively inhibiting synthesis of downstream sterol components has no effect on infection, suggesting that the effect of statins on host nonsterol intermediates is detrimental to bacterial growth. Furthermore, statins also reduce bacterial proliferation in the S. enterica serovar Typhimurium mouse model. This suggests that blocking the production of nonsterol precursors in the host cell can be used to reduce infection.

The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55

Intracellular, pathogenic Salmonella typhimurium avoids phago‐lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella‐containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI‐2). Furthermore, the construction of a three‐dimensional space‐filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol‐rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)‐anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol‐rich early endocytic pathways during intracellular bacterial replication.

The affinity of cholesterol for phosphatidylcholine and sphingomyelin.

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for N-palmitoyl sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.

Cell cholesterol homeostasis: Mediation by active cholesterol

Recent evidence suggests that the major pathways mediating cell cholesterol homeostasis respond to a common signal: active membrane cholesterol. Active cholesterol is the fraction that exceeds the complexing capacity of the polar bilayer lipids. Increments in plasma membrane cholesterol exceeding this threshold have an elevated chemical activity (escape tendency) and redistribute via diverse transport proteins to both circulating plasma lipoproteins and intracellular organelles. Active cholesterol thereby prompts several feedback responses. It is the substrate for its own esterification and for the synthesis of regulatory side-chain oxysterols. It also stimulates manifold pathways that down-regulate the biosynthesis, curtail the ingestion and increase the export of cholesterol. Thus, the abundance of cell cholesterol is tightly coupled to that of its polar lipid partners through active cholesterol.