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Four cholesterol-sensing proteins
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Four cholesterol-sensing proteins Yvonne Lange* and Theodore L Steckt What is the connection among the following three medical conditions: Niemann-Pick type C disease (a cause of mental retardation and early death), systemic lipidosis (in which an obscure side effect of numerous drugs transforms lysosomes into lamellar bodies), and holoprosencephaly (a catastrophe in embryonic development)? Recent evidence suggests that the pathogenesis in each case involves impaired sensing of cellular cholesterol. Addresses
*Department of Pathology, Rush Presbyterian St Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, USA; e-mail: [email protected] tDepartment of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 6063?, USA; e-mail: [email protected] Current Opinion in Structural Biology 1998, 8:435-439
http://biomednet,comletecref/Og59440XO0800435 © Current Biology Publications ISSN 0959-440X Abbreviations ACAT ER NPC SCAP SREBP SSD StAR protein
acyI-CoA:cholesterol acyltransferase endoplasmic reticulum Niemann-Pick type C SREBP cleavage-activating protein sterol regulatory element-binding protein sterol-sensing domain steroidogenic acute regulatory protein
Introduction Sterols appear to have evolved to fill the flickering spaces among the fatty acyl chains in fluid membrane bilayers [1,2]. This condenses the bilayer, reducing its passive permeability and increasing its mechanical durability and rigidity. It is not surprising, therefore, that sterols are ubiquitous among eukaryotes and are particularly plentiful in the plasma membrane. Membrane sterols vary among organisms, although the cholesterol found in animal cells represents the family well. This review will consider four integral membrane proteins that apparently respond to cholesterol signals. T h e s e proteins share a sequence motif, which spans five consecutive transmembranc strands, thought to function as a sterolsensing domain (SSD) (Figure 1) [3,4"]. It seems that perturbations of SSD-bearing proteins underlie the disparate medical conditions mentioned above. Of course, the sensing of intracellular cholesterol is also highly relevant to the pathophysiology of a major killer, atherosclerosis.
Intracellular cholesterol dynamics Cells obtain cholesterol primarily through its biosynthesis in the endoplasmic reticulum (ER) and its release from ingested low density lipoproteins in lysosomes. Cholesterol beyond the cell's needs is either stored in cytoplasmic ester
droplets, exported from the cell surface or, in the mitochondria and/or ER of specialized cells, converted to steroid hormones, bile salts or serum lipoproteins. Despite their insolubility in water, sterols move briskly around the cell. Nascent sterols are transported from the ER to the plasma membrane within minutes [5,6"]. T h e entire plasma membrane cholesterol pool fluxes through the ER in about an hour [7] and the lysosomal cholesterol pool empties into the plasma membrane in less than an hour [8,9]. Lysosomal cholesterol is transported to the ER, either directly [10] or indirectly, via the plasma membrane [11]. Plasma membrane cholesterol also moves to mitochondria [12,13] and, apparently, to the Golgi apparatus [14]. T h e balance of these fluxes is such that plasma membranes (and their endocytic derivatives) contain roughly 10 times more cholesterol than the rest of the membranes combined [15,16]. Presumably, it is also this vectorial circulation that allocates most of the late sterol intermediates to the plasma membrane, even though they are generated and utilized in the ER [5]. At least some cholesterol moves through endomembrane pathways. This route could account for all of the transfer of plasma membrane cholesterol to the lysosomes, which proceeds at about 5% per hour [9]. In addition, cholesterol associates with sphingolipids in distal Golgi membranes, forming functional microdomains called rafts [17]. These structures presumably arise by lateral phase separation - a distinctive form of topographic information and organization [18"]. Rafts appear to escort and perhaps target certain proteins to the cell surface [19",20"]. T h e importance of rafts as vehicles for cholesterol transport is presently unclear. Similarly, while there is evidence that small vesicles shuttle sterols through the cell, these membranes have yet to be isolated and characterized [16]. Over the years, certain water-soluble cellular proteins have been found to facilitate the intermembrane transfer of sterols in vitro [21"]. It now appears that the hydrophobic binding pockets of these 'sterol-carrier proteins' actually serve other physiological functions (see, for an example, [22"]). In contrast, the cholesterol-binding protein caveolin is clearly associated with the caveolae - - s t e r o l - r i c h , muhifunctional plasma m e m b r a n e invaginations [17,23]. Despite the presence of an apparent hydrophobic anchor, a small fraction of caveolin may form soluble protein complexes that serve to transport intracellular cholesterol to the plasma membrane [6"]. Also, caveolae may be the site at which cholesterol is transferred in both directions between the cell surface and plasma high-density lipoproteins, via the scavenger receptor, SR-BI [21",24"[. Furthermore, supporting a physiological role for caveolae in cholesterol homeostasis
436 Lipids
Figure I
(a)
(b)
HMG-CoA reductase
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I
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Four cholesterol-sensing proteins. Some organizational similarities are shown for four integral membrane proteins thought to respond to cholesterol. (a) Patched, (b) HMG-CoA reductase, (¢) Niemann-Pick type C protein 1 (NPC1) and (d) SOAP. The five helical stretches shown in boxes represent sequence-related putative sterol-sensing domains (SSDs). The darkened transmembrane helices in Patched and NPC1 highlight extended sequence similarities. Reproduced from [4"*] with permission.
is the influence that cellular cholesterol exerts over caveolin gene expression [25",26"].
transcription factors that control the expression of other regulatory elements [28,29"'].
T h e provision of plasma membrane cholesterol to the matrix space of mitochondria is required for steroidogenesis [12]. In order to reach the oxidative machinery, incoming cholesterol must be transported across both mitochondrial membranes, perhaps at their junction. T h e newly described steroidogenic acute regulatory (STAR) protein facilitates this transport process [23,27"], although whether the StAR protein has sterol-binding or transport activity remains to be established.
It may be that all of these homeostatic proteins are orchestrated by- a common regulatory signal - - the level of ER cholesterol in their vicinity [13,30,31"']. This is clearly the case for ACAT, the catalytic activity of which is acutely dependent on local cholesterol concentrations [32,33"]. In contrast, it is primarily the abundance of HMG-CoA reductase (the initial control point in the pathway of sterol biosynthesis), rather than its catalytic constant, that is governed by cellular cholesterol levels. Protein turnover is one of several ways that HMG-CoA reductase is controlled; its susceptibility to proteolysis is increased by cholesterol, perhaps through the SSD motif located in its intramembrane domain [3,34,35].
Intracellular cholesterol homeostasis Cellular cholesterol levels are controlled by a diverse set of homeostatic activities that are located primarily in the ER. T h e s e include enzymes for sterol biosynthesis (e.g. HMG-CoA reductase) and esterification (acyI-CoA:cholesterol acyhransferase, ACAT), as well as precursors for
T h e expression of genes for the low-density lipoprotein receptor, HMG-CoA reductasc and other regulators of
Fourcholesterol-sensingproteinsLangeand Steck
cholesterol abundance is under the positive control of a family of transcription factor precursors called sterol regulatory element-binding proteins (SREBPs) [29"]. When cellular cholesterol levels fall, the active domain of the SREBPs is solubilized from the ER by proteolysis and translocated to the nucleus. The first and pivotal of the two proteolytic cuts in SREBP is under the control of SCAP [35,36], the SREBP cleavage-activating protein with which it is complexed [37,38"]. An SSD domain within SCAP apparently transduces the reduction in the ER cholesterol level into a signal that activates the requisite proteases. For example, when the level of cholesterol in the ER is increased by treating cells with sphingomyelinase [31"], SCAP activity is reduced [39"]. SREBPs also exert negative control on caveolin gene expression [25"]; the stimulation of caveolin synthesis by cholesterol is presumably homeostatic because caveolae mediate cholesterol export [21",24"]. How is the level of ER cholesterol adjusted physiologically in order to play this regulatory role? We estimate that the ER pool contains only -0.1-2.0% of the total cellular cholesterol [31"], its level being set by the stream of plasma membrane cholesterol flowing through it [30]. This circulation could be regulated to set the level of cholesterol in the ER compartment according to the needs of the plasma membrane. Since modest changes in the plasma membrane pool elicit large responses in the ER, there would appear to be a cholesterol sensor with a threshold [13,30].
Lipidosis T h e accumulation of membrane lipids in lamellar bodies is a widely observed side effect of dozens of amphiphilic drugs, experimental agents and natural products [40,41]. Lamellar bodies are lysosome-like organelles that are filled with cholesterol-rich, densely packed membranous whorls [42]. The agents causing this side effect, which are called class 2 amphiphiles, arc mostly hydrophobic amines, although progesterone and other steroids are also prominent members of this diverse group [13,42--46]. T h e untoward action of class 2 amphiphiles could be to reduce the ER cholesterol pool perhaps by blocking the flux of the plasma membrane cholesterol to the ER [13,30]. As a result, the various homeostatic elements in the ER would drive cholesterol accretion through misguided homeostatic responses [30,44,47]. Sterols would then accumulate to excess in the Golgi apparatus [43], plasma membranes (Y Lange, T L Steck, unpublished data) and lysosomes [40]. The formation of such lamellar bodies could represent the cell's attempt to dispose of the excess lipid [41]. The amphiphiles would also cause the accumulation of sterol intermediates, if their return from the plasma membrane to the ER were inhibited. This would reduce the biosynthesis of cholesterol in the face of increased sterol production and accumulation [13,45--47]. It is of great interest that the potency of numerous amphiphiles as inhibitors of cholesterol delivery to the ER
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correlates well with their effect on multidrug resistance transport activity, since it raises the possibility of a mechanistic role for P-glycoproteins in cholesterol transport [23,46,48].
Niemann-Pick type C disease Niemann-Pick type C (NPC) disease is a rare homozygous recessive disorder that leads to mental retardation, complications in multiple organ systems and early death [49]. The affected gene, NPC1, encodes a putative integral membrane glycoprotein, with an SSD [50",51] whose subcellular disposition has not yet been determined. NPC1 is disrupted in diverse ways in the mutant alleles thus far examined [50",51 ]. The NPC phenotype is notably similar to that of cells treated with class 2 amphiphiles. Both feature massive accumulation of cholesterol in lysosomal lamellar bodies [41] and both exhibit alterations in sterol homeostasis that are consistent with low levels of cholesterol in the ER [49,52]. The NPC1 protein could therefore be the site of action of the amphiphiles.
Holoprosencephaly This term refers to a set of major developmental deformities affecting the midline of the brain and face - - classically marked by a cyclopic eye. There is diverse evidence for the fact that a disturbance of fetal cholesterol metabolism could be involved in some cases [53]. Three surprising findings have now shed light on this connection [4"]. The first surprise is that the morphogenetic signal protein Hedgehog is anchored at the cell surface through a novel ester linkage between its terminal carboxyl function and the hydroxyl group of cholesterol. Notably, this modification is created by Hedgehog autoprocessing [54]. The purpose of this tether ,night be to restrict the diffusion of the morphogen in a way that other forms of membrane integration cannot accomplish [4"]. It was also a surprise that the target for Hedgehog signaling, an integral membrane protein called Patched, bears an overall similarity to NPC1, including an SSD sequence like those seen in the cholesterol homeostasis proteins (Figure 1) [50"]. The question then arises as to whether the mechanistic link between cholesterol and holoprosencephaly occurs through the Hedgehog adduct or the SSD on Patched [4"]. The third surprise is that steroidal amines from certain plants, previously known to elicit holoprosencephaly, were also found to perturb cholesterol homeostasis in the manner of class 2 amphiphiles ([4"',55"']; PA Beachy, personal communication). That is, they inhibit cholesterol esterification and cause the accumulation of late sterol intermediates. Conversely, various unrelated class 2 amphiphiles were found to mimic these plant alkaloids in an in vitro developmental system. Since the Hedgehog protein was normal in these experiments, it is an attractive possibility that the teratogenic alkaloids act in vivo as class 2
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amphiphiles, undermining Patched function through a direct or an indirect effect upon its SSD [55"°].
Conclusions Four distinctly different proteins, NPC1, HMG-CoA reductase, SCAP and Patched, share a membranespanning sequence motif that is suggested to function as a SSD. What we now need is direct biochemical and structural information as to whether SSD motifs actually sense and/or signal sterol levels, how they might do so and how class 2 amphiphiles might perturb these activities. Some guiding hypotheses concerning the physiology and pathophysiology of these proteins follow. NPC 1 might serve as a sensor of the abundance of plasma membrane cholesterol and set the ER cholesterol pool size accordingly. The defect in NPC1 mutations and cells that have been exposed to class 2 amphiphiles could reduce ER cholesterol to an inappropriately low level, for example, by perturbing either the sensor threshold mechanism or a subsequent transport step. The physiological response, a build up of cholesterol and its precursors, would lead to secondary effects, such as lamellar-body formation and cellular injury. The chain of causality in this model runs opposite to that of the reigning paradigm, in which a primary lysosomal transport lesion results in impaired ER cholesterol metabolism [10,43,49]. Precision in cell cholesterol homeostasis could be enhanced by a two-tiered system of control - - the coarse adjustments made in the size of the regulatory ER pool by the upstream SSD-based sensor, NPC1, and the fine tuning achieved by the cholesterol-sensing elements in the ER. The latter proteins include ACAT, which apparently lacks an SSD, and the SSD-bearing proteins HMG-CoA reductase and SCAR Some forms of holoprosencephaly could arise from direct interactions between class 2 amphiphiles and the SSD on Patched. Alternatively, these malformations could be secondary to the aberrant sterol metabolism induced by these agents at a critical point in morphogenesis.
Acknowledgements We arc grateful to PA Beachy for providing unpublished findings and a copy of 155°° ] prior to its publication.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ° of outstanding interest 1.
Bloom M, Evans E, Mouritsen O: Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q Rev Biophys 1991, 24:293-397.
2.
Mouritsen OG, Jorgensen K: Dynamical order and disorder in lipid bilayers. Chem Phys Lipids 1994, 73:3-25.
3.
Hua X, Nohturfft A, Goldstein JL, Brown M: Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein. Ceil 1996, 87:415-426.
4. •,
Beachy PA, Cooper MK, Young KE, yon Kessler DP, Park W-J, Tanaka Hall TM, Leahy DJ, Porter J: Multiple roles of cholesterol in hedgehog protein biogenesis and signaling. Cold Spring Harb Syrup Quant Biol 1997, 62:191-204. Multiple connections are noted between cholesterol and early embryonic development. First is the tethering of a morphogenetic signal protein (Hedgehog) to the cell surface through a novel covalent bond to cholesterol. Secondly, an integral membrane protein (Patched), which is responsive to Hedgehog signals, has a putative sterol-sensing domain. Third, certain teratogens perturb cholesterol homeostasis. 5.
Lange Y, Echevarria F, Steck TL: Movement of zymosterol, a precursor of cholesterol, among three membranes in human fibroblasts. J Biol Chem 1991,266:21439-21443.
6. •e
Uittenbogaard A, Ying Y-s, Smart EJ: Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. Involvement in cholesterol trafficking. J Biol Chem 1998, 273:6525-6532. A fraction of caveolin is reported to form a soluble immunophilin-chaperone complex, which stimulates the movement of intracellular cholesterol to the plasma membrane. 7.
Lange Y, Strebel F, Steck TL: Role of the plasma membrane in cholesterol esterification in rat hepatoma cells. J Biol Chem 1993, 268:13838-13843.
8.
Brasaemle DL, Attie AD: Rapid intracellular transport of LDLderived cholesterol to the plasma membrane in cultured fibroblasts. J Lipid Res 1990, 31:103-112.
9.
Lange Y, Ye J, Steck TL: CirculaUon of cholesterol between lysosomes and the plasma membrane. J Bio/Chem 1998, 273:18915-18922.
10. Underwood KW, Jacobs NL, Howley A, Liscum L: Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane. J Bio/ Chem 1998, 273:4266-4274. 11. Lange Y, Ye J, Chin J: The fate of cholesterol exiting lysosomes. J Bio/Chem 1997, 272:17018-17022. 12. Freeman DA: Plasma membrane cholesterol: removal and insertion into the membrane and utilization as substrate for steroidogenesis. Endecrino/ogy 1989, 124:2527-2534. 13. Lange Y, Steck TL: Cholesterol homeostasis. Modulation by amphiphiles. J Bio/Chem 1994, 269:29371-29374. 14. Conrad PA, Smart El, Ying YS, Anderson RG, Bloom G: Caveolin cycles between plasma membrane caveolae and the Golgi complex bymicrotubule-dependent and microtubule-independent steps. J Ceil Bio/1995, 131:1421-1433. 15. Lange Y, Swaisgood MH, Ramos B, Steck TL: Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J Bio/Chem 1989, 264:3786-3793. 16. Liscum L, Underwood K: Intracellular cholesterol transport and compartmentation. J Bio/Chem 1995, 270:15443-15446. 17,
Harder T, Simons K: Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr Opin Ceil Bio/1997, 9:534-542.
18. Brown DA, London E: Structure of detergent-resistant membrane • domains: does phase separation occur in biological membranes? Biochem Biophys Res Commun 1997, 240:1-7. The significance of cholesteroFrich membrane bilayer domains that are stable in nonionic detergents is critically evaluated. 19. Scheiffele P, Verkade P, Fra AM, Virta H, Simons K, Ikonen E: • Caveolin-1 and -2 in the exocytic pathway of MDCK cells. J Ce// Biol 1998, 140:795-806. This paper provides evidence for both the vectorial movement of nascent caveolin isoforms and their role in targeting proteins to the surfaces of polarized epithelial cells. 20. Keller P, Simons K: Cholesterol is required for surface transport of he influenza virus hemaggliutinin. J Ceil Bio/1998, 140:137-1367. fact that cholesterol-sphingolipid rafts are vehicles in targeted exocytosis traffic is persuasively documented. 21. Fielding C J, Fielding PE: Intracellular cholesterol transport. J Lipid A Res 1997, 38:1503-1521. broad review, featuring the mechanisms that mediate cholesterol import from and export to high density lipoproteins. 22. Antonenkov V, Van Veldhoven P, Waelkens E, Mannaerts G: Substrate • specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyI-CoA thiolase purified from normal rat liver
Four cholesterol-sensing proteins Lange and Steck
peroxisomes. Sterol carrier protein 2/3-oxoacyI-CoA thiolase is
involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates. J Biol Chem 199?, 272:26023-26031. A strong contribution to the growing evidence suggesting that so-called 'sterol carrier proteins' may not serve this function in vivo. 23. Ikonen E: Molecular mechanisms of intracellular cholesterol transport. Curt Opin Lipido/1997, 8:60-64. 24. Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RG, Xu S, Krieger M: • Murine SR-BI, a high density lip•protein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J Bio/Chem 199?, 272:13242-13249. Caveolae are sterol-rich membrane domains that appear to carry out multiple structural and functional roles at the cell surface and in the cell interior. SR-BI, the receptor that mediates the transfer of cholesterol between plasma membranes and high-density plasma lip•proteins and therefore plays an important role in both cell and whole-body cholesterol homeostasis, is shown to be concentrated in caveolae. 25. Bist A, Fielding PE, Fielding C: Two sterel regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lip•protein free cholesterol. Proc Nat/ Acad Sci USA 1997, 94:10693-10698. Cholesterol is shown to positively regulate the expression of caveolin.
•
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from in vivo competition studies. J Bio/ Chem 1998, 273:5765-5793. SREBPs are transcription factors that are activated by cleavage from the endoplasmic reticulum (ER) membrane under cholesterol control. The ER protein SCAP (SREBP cleavage-activating protein) is thought to regulate this proteolytic process, presumably using its sterol-sensing domain. This study provides evidence for the direct association of SCAP with its target, the SREBPs. 39. Scheek S, Brown MS, Goldstein J: Sphingomyelin depletion in • cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1. Proc Nat/Acad Sci USA 1997, 94:11179-11183.
The authors describe direct evidence that the proteolytic activation of SREBP is made in response to ER levels of cholesterol. 40. Hruban Z: Pulmonary and generalized lysosomal storage induced by amphiphilic drugs. Environ Health Perspect 1984, 55:53-76. 41. Schmitz G, Muller G: Structure and function of lamellar bodies, lipid-protein complexes involved in storage and secretion of cellular lipids. J Lipid Res 1991, 32:1539-1570. 42. McGookey DJ, Anderson R: Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells. J Cell Bio/1983, 97:1156-1166.
26. Hailstones D, Sleet LS, Parton RG, Stanley K: Regulation of caveolin • and caveolae by cholesterol in MDCK cells. J Lipid Res 1998, 39:369-379. Cholesterol is shown to positively regulate the expression of caveolin.
43. Buffer JD, Blanchette-Mackie J, Gotdin E, O'Neill RR, Carstea G, Roff CF, Patterson MC, Patei S, Cornly ME, Cooney A e t al.: Progesterone blocks cholesterol translocation from lysosomes. J Biol Chem 1992, 267:23797-23805.
27. Stocco D: A review of the characteristics of the protein required • for the acute regulation of steroid hormone biosynthesis: the case for the steroidogenic acute regulatory (STAR) protein. Proc Soc Exp Bio/ Med 1998, 217:123-129. An up-to-date overview of a newly discovered mitochondrial protein that regulates steroid•genesis.
44. MazzoneT, Krishna M, Lange Y: Progesterone blocks intracellular translocation of free cholesterol derived from cholesterol ester in macrophages. J Lipid Res 1995, 36:544-551.
28. Goldstein JL, Brown M: Regulation of the mevalonate pathway. Nature 1990, 343:425-430.
46. Metherall JE, Li H, Waugh K: Role of multidrug resistance Pglycoproteins in cholesterol biosynthesis. J Biol Chern 1996, 271:2634-2640.
29. Brown MS, Goldstein JL: The SREBP pathway: regulation of 00 cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Ceil 1997, 89:331-340. An overview of the system by which ER transcription factors regulate the expression of ER proteins that mediate cholesterol homeostasis. 30. Lange Y, Steck TL: The role of intracellular cholesterol transport in cholesterol homeostasis. Trends Ceil Bio/1996, 6:205-208. 31. Lange Y, Steck TL: Quantification of the pool of cholesterol • • associated with acyI-CoA cholesterol acyltransferase in human fibroblasts. J Bio/Chem 199?, 272:13103-13108. Most of the proteins that contol the level of cholesterol in cells are located in the endoplasmic reticulum (ER). The pool of cholesterol in the ER membrane is shown to be very small and very responsive to physiological stimuli. The size of the ER cholesterol compartment therefore appears to be regulated so as to serve as a critical element in cholesterol homeostasis. 32. Cheng D, Chang CC, Clu X, Chang T: Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system. J Bio/Chem 1995, 270:685-695. 33. Chang TY, Chang CC, Cheng D: Acyl-coenzyme A:cholesterol acyltransferase. Annu Rev Biochem 1997, 66:613-638. A definitive review of a critical element in cholesterol homeostasis. 34. •lender EH, Simoni R: The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase. J Bio/ Chem 1992, 267:4223-4235. 35. McGee TP, Cheng HH, Kumagai H, Ornura S, Sirnoni R: Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis. J Bio/Chem 1996, 271:25630-25638. 36. Sakai J, Duncan EA, Rawson RB, Hua X, Brown MS, Goldstein J: Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment. Ceil 1996, 85:1037-1046. 37. Sakai J, Nohturfft A, Cheng D HY, Brown MS, Goldstein J: Identification of complexes between the CO•H-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein. J Bio/Chem 1997, 272:20213-20221. 38. Sakai J, Nohturfft A, Goldstein JL, Brown MS: Cleavage of sterol • regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence
45. Metherall JE, Waugh K, Li H: Progesterone inhibits cholesterol biosynthesis in cultured cells. Accumulation of cholesterol precursors. J Biol Chem 1996, 271:2627-2633.
4?.
FieldFJ, Born E, Murthy S, Mathur S: Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in CaCo-2 cells and differs from the transport of
plasma membrane cholesterol to the endoplasmic reticulum. J Lipid Res 1998, 39:333-343. 48. Debry P, Nash EA, Neklason DW, Metherall J: Role of multidrug resistance P-glycoproteins in cholesterol esterification. J Biol Chem 1997, 272:1026-1031. 49. Pentchev PG, Vanier MT, Suzuki K, Patterson MC: Niemann-Pick disease type C: a cellular cholesterol lipid•sis. In The Metabofic and Molecular Bases of Inherited Disease. Edited by Scriver CR, Beaudet AL, Sly WS, Valle D, Stanbury JB, Wyngaarden JB, Fredrickson DS. New York: McGraw-Hill; 1995:2625-2639. 50. Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings 00 C, Gu J, Rosenfeld MA, Pavan WJ, Krizman DB et aL: Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 199"7, 277:228-231. The identification of NPC1, the gene for a fatal cholesterol storage disease, suggests that it is an integral membrane glycoprotein with a sterol-sensing domain. 51. Loftus SK, Morris JA, Carstea ED, Gu JZ, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld MA, Tagle DA et al.: Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 199?, 277:232-235. 52. Pentchev PG, Brady RO, Blanchette-Mackie El, Vanier MT, Carstea ED, Parker CC, Goldin E, Roff CF: The Niemann-Pick C lesion and its relationship to the intracellular distribution and utilization of LDL cholesterol. Biochim Biophys Acta 1994, 1225:235-243. 53. Herz J, Willnow TE, Farese RJ: Cholesterol, hedgehog and embryogenesis. Nat Genet 199?, 15:123-124. 54. Porter JA, Young KE, Beachy P: Cholesterol modification of hedgehog signaling proteins in animal development. Science 1996, 274:255-259. 55. Cooper MK, Porter JA, Young KE, Beachy P: Teratogen-mediated 00 inhibition of target tissue response in Sonic hedgehog signaling. Science 1998, 280:1603-1607. Amphiphiles that perturb sterol homeostasis affect the morphogenetic control system, which responds to Hedgehog. The target of these amphiphiles may be Patched, a sterol-sensing receptor for Hedgehog.
Four cholesterol-sensing proteins Yvonne Lange* and Theodore L Steckt What is the connection among the following three medical conditions: Niemann-Pick type C disease (a cause of mental retardation and early death), systemic lipidosis (in which an obscure side effect of numerous drugs transforms lysosomes into lamellar bodies), and holoprosencephaly (a catastrophe in embryonic development)? Recent evidence suggests that the pathogenesis in each case involves impaired sensing of cellular cholesterol. Addresses
*Department of Pathology, Rush Presbyterian St Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, USA; e-mail: [email protected] tDepartment of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 6063?, USA; e-mail: [email protected] Current Opinion in Structural Biology 1998, 8:435-439
http://biomednet,comletecref/Og59440XO0800435 © Current Biology Publications ISSN 0959-440X Abbreviations ACAT ER NPC SCAP SREBP SSD StAR protein
acyI-CoA:cholesterol acyltransferase endoplasmic reticulum Niemann-Pick type C SREBP cleavage-activating protein sterol regulatory element-binding protein sterol-sensing domain steroidogenic acute regulatory protein
Introduction Sterols appear to have evolved to fill the flickering spaces among the fatty acyl chains in fluid membrane bilayers [1,2]. This condenses the bilayer, reducing its passive permeability and increasing its mechanical durability and rigidity. It is not surprising, therefore, that sterols are ubiquitous among eukaryotes and are particularly plentiful in the plasma membrane. Membrane sterols vary among organisms, although the cholesterol found in animal cells represents the family well. This review will consider four integral membrane proteins that apparently respond to cholesterol signals. T h e s e proteins share a sequence motif, which spans five consecutive transmembranc strands, thought to function as a sterolsensing domain (SSD) (Figure 1) [3,4"]. It seems that perturbations of SSD-bearing proteins underlie the disparate medical conditions mentioned above. Of course, the sensing of intracellular cholesterol is also highly relevant to the pathophysiology of a major killer, atherosclerosis.
Intracellular cholesterol dynamics Cells obtain cholesterol primarily through its biosynthesis in the endoplasmic reticulum (ER) and its release from ingested low density lipoproteins in lysosomes. Cholesterol beyond the cell's needs is either stored in cytoplasmic ester
droplets, exported from the cell surface or, in the mitochondria and/or ER of specialized cells, converted to steroid hormones, bile salts or serum lipoproteins. Despite their insolubility in water, sterols move briskly around the cell. Nascent sterols are transported from the ER to the plasma membrane within minutes [5,6"]. T h e entire plasma membrane cholesterol pool fluxes through the ER in about an hour [7] and the lysosomal cholesterol pool empties into the plasma membrane in less than an hour [8,9]. Lysosomal cholesterol is transported to the ER, either directly [10] or indirectly, via the plasma membrane [11]. Plasma membrane cholesterol also moves to mitochondria [12,13] and, apparently, to the Golgi apparatus [14]. T h e balance of these fluxes is such that plasma membranes (and their endocytic derivatives) contain roughly 10 times more cholesterol than the rest of the membranes combined [15,16]. Presumably, it is also this vectorial circulation that allocates most of the late sterol intermediates to the plasma membrane, even though they are generated and utilized in the ER [5]. At least some cholesterol moves through endomembrane pathways. This route could account for all of the transfer of plasma membrane cholesterol to the lysosomes, which proceeds at about 5% per hour [9]. In addition, cholesterol associates with sphingolipids in distal Golgi membranes, forming functional microdomains called rafts [17]. These structures presumably arise by lateral phase separation - a distinctive form of topographic information and organization [18"]. Rafts appear to escort and perhaps target certain proteins to the cell surface [19",20"]. T h e importance of rafts as vehicles for cholesterol transport is presently unclear. Similarly, while there is evidence that small vesicles shuttle sterols through the cell, these membranes have yet to be isolated and characterized [16]. Over the years, certain water-soluble cellular proteins have been found to facilitate the intermembrane transfer of sterols in vitro [21"]. It now appears that the hydrophobic binding pockets of these 'sterol-carrier proteins' actually serve other physiological functions (see, for an example, [22"]). In contrast, the cholesterol-binding protein caveolin is clearly associated with the caveolae - - s t e r o l - r i c h , muhifunctional plasma m e m b r a n e invaginations [17,23]. Despite the presence of an apparent hydrophobic anchor, a small fraction of caveolin may form soluble protein complexes that serve to transport intracellular cholesterol to the plasma membrane [6"]. Also, caveolae may be the site at which cholesterol is transferred in both directions between the cell surface and plasma high-density lipoproteins, via the scavenger receptor, SR-BI [21",24"[. Furthermore, supporting a physiological role for caveolae in cholesterol homeostasis
436 Lipids
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D
C
Four cholesterol-sensing proteins. Some organizational similarities are shown for four integral membrane proteins thought to respond to cholesterol. (a) Patched, (b) HMG-CoA reductase, (¢) Niemann-Pick type C protein 1 (NPC1) and (d) SOAP. The five helical stretches shown in boxes represent sequence-related putative sterol-sensing domains (SSDs). The darkened transmembrane helices in Patched and NPC1 highlight extended sequence similarities. Reproduced from [4"*] with permission.
is the influence that cellular cholesterol exerts over caveolin gene expression [25",26"].
transcription factors that control the expression of other regulatory elements [28,29"'].
T h e provision of plasma membrane cholesterol to the matrix space of mitochondria is required for steroidogenesis [12]. In order to reach the oxidative machinery, incoming cholesterol must be transported across both mitochondrial membranes, perhaps at their junction. T h e newly described steroidogenic acute regulatory (STAR) protein facilitates this transport process [23,27"], although whether the StAR protein has sterol-binding or transport activity remains to be established.
It may be that all of these homeostatic proteins are orchestrated by- a common regulatory signal - - the level of ER cholesterol in their vicinity [13,30,31"']. This is clearly the case for ACAT, the catalytic activity of which is acutely dependent on local cholesterol concentrations [32,33"]. In contrast, it is primarily the abundance of HMG-CoA reductase (the initial control point in the pathway of sterol biosynthesis), rather than its catalytic constant, that is governed by cellular cholesterol levels. Protein turnover is one of several ways that HMG-CoA reductase is controlled; its susceptibility to proteolysis is increased by cholesterol, perhaps through the SSD motif located in its intramembrane domain [3,34,35].
Intracellular cholesterol homeostasis Cellular cholesterol levels are controlled by a diverse set of homeostatic activities that are located primarily in the ER. T h e s e include enzymes for sterol biosynthesis (e.g. HMG-CoA reductase) and esterification (acyI-CoA:cholesterol acyhransferase, ACAT), as well as precursors for
T h e expression of genes for the low-density lipoprotein receptor, HMG-CoA reductasc and other regulators of
Fourcholesterol-sensingproteinsLangeand Steck
cholesterol abundance is under the positive control of a family of transcription factor precursors called sterol regulatory element-binding proteins (SREBPs) [29"]. When cellular cholesterol levels fall, the active domain of the SREBPs is solubilized from the ER by proteolysis and translocated to the nucleus. The first and pivotal of the two proteolytic cuts in SREBP is under the control of SCAP [35,36], the SREBP cleavage-activating protein with which it is complexed [37,38"]. An SSD domain within SCAP apparently transduces the reduction in the ER cholesterol level into a signal that activates the requisite proteases. For example, when the level of cholesterol in the ER is increased by treating cells with sphingomyelinase [31"], SCAP activity is reduced [39"]. SREBPs also exert negative control on caveolin gene expression [25"]; the stimulation of caveolin synthesis by cholesterol is presumably homeostatic because caveolae mediate cholesterol export [21",24"]. How is the level of ER cholesterol adjusted physiologically in order to play this regulatory role? We estimate that the ER pool contains only -0.1-2.0% of the total cellular cholesterol [31"], its level being set by the stream of plasma membrane cholesterol flowing through it [30]. This circulation could be regulated to set the level of cholesterol in the ER compartment according to the needs of the plasma membrane. Since modest changes in the plasma membrane pool elicit large responses in the ER, there would appear to be a cholesterol sensor with a threshold [13,30].
Lipidosis T h e accumulation of membrane lipids in lamellar bodies is a widely observed side effect of dozens of amphiphilic drugs, experimental agents and natural products [40,41]. Lamellar bodies are lysosome-like organelles that are filled with cholesterol-rich, densely packed membranous whorls [42]. The agents causing this side effect, which are called class 2 amphiphiles, arc mostly hydrophobic amines, although progesterone and other steroids are also prominent members of this diverse group [13,42--46]. T h e untoward action of class 2 amphiphiles could be to reduce the ER cholesterol pool perhaps by blocking the flux of the plasma membrane cholesterol to the ER [13,30]. As a result, the various homeostatic elements in the ER would drive cholesterol accretion through misguided homeostatic responses [30,44,47]. Sterols would then accumulate to excess in the Golgi apparatus [43], plasma membranes (Y Lange, T L Steck, unpublished data) and lysosomes [40]. The formation of such lamellar bodies could represent the cell's attempt to dispose of the excess lipid [41]. The amphiphiles would also cause the accumulation of sterol intermediates, if their return from the plasma membrane to the ER were inhibited. This would reduce the biosynthesis of cholesterol in the face of increased sterol production and accumulation [13,45--47]. It is of great interest that the potency of numerous amphiphiles as inhibitors of cholesterol delivery to the ER
437
correlates well with their effect on multidrug resistance transport activity, since it raises the possibility of a mechanistic role for P-glycoproteins in cholesterol transport [23,46,48].
Niemann-Pick type C disease Niemann-Pick type C (NPC) disease is a rare homozygous recessive disorder that leads to mental retardation, complications in multiple organ systems and early death [49]. The affected gene, NPC1, encodes a putative integral membrane glycoprotein, with an SSD [50",51] whose subcellular disposition has not yet been determined. NPC1 is disrupted in diverse ways in the mutant alleles thus far examined [50",51 ]. The NPC phenotype is notably similar to that of cells treated with class 2 amphiphiles. Both feature massive accumulation of cholesterol in lysosomal lamellar bodies [41] and both exhibit alterations in sterol homeostasis that are consistent with low levels of cholesterol in the ER [49,52]. The NPC1 protein could therefore be the site of action of the amphiphiles.
Holoprosencephaly This term refers to a set of major developmental deformities affecting the midline of the brain and face - - classically marked by a cyclopic eye. There is diverse evidence for the fact that a disturbance of fetal cholesterol metabolism could be involved in some cases [53]. Three surprising findings have now shed light on this connection [4"]. The first surprise is that the morphogenetic signal protein Hedgehog is anchored at the cell surface through a novel ester linkage between its terminal carboxyl function and the hydroxyl group of cholesterol. Notably, this modification is created by Hedgehog autoprocessing [54]. The purpose of this tether ,night be to restrict the diffusion of the morphogen in a way that other forms of membrane integration cannot accomplish [4"]. It was also a surprise that the target for Hedgehog signaling, an integral membrane protein called Patched, bears an overall similarity to NPC1, including an SSD sequence like those seen in the cholesterol homeostasis proteins (Figure 1) [50"]. The question then arises as to whether the mechanistic link between cholesterol and holoprosencephaly occurs through the Hedgehog adduct or the SSD on Patched [4"]. The third surprise is that steroidal amines from certain plants, previously known to elicit holoprosencephaly, were also found to perturb cholesterol homeostasis in the manner of class 2 amphiphiles ([4"',55"']; PA Beachy, personal communication). That is, they inhibit cholesterol esterification and cause the accumulation of late sterol intermediates. Conversely, various unrelated class 2 amphiphiles were found to mimic these plant alkaloids in an in vitro developmental system. Since the Hedgehog protein was normal in these experiments, it is an attractive possibility that the teratogenic alkaloids act in vivo as class 2
438
Lipids
amphiphiles, undermining Patched function through a direct or an indirect effect upon its SSD [55"°].
Conclusions Four distinctly different proteins, NPC1, HMG-CoA reductase, SCAP and Patched, share a membranespanning sequence motif that is suggested to function as a SSD. What we now need is direct biochemical and structural information as to whether SSD motifs actually sense and/or signal sterol levels, how they might do so and how class 2 amphiphiles might perturb these activities. Some guiding hypotheses concerning the physiology and pathophysiology of these proteins follow. NPC 1 might serve as a sensor of the abundance of plasma membrane cholesterol and set the ER cholesterol pool size accordingly. The defect in NPC1 mutations and cells that have been exposed to class 2 amphiphiles could reduce ER cholesterol to an inappropriately low level, for example, by perturbing either the sensor threshold mechanism or a subsequent transport step. The physiological response, a build up of cholesterol and its precursors, would lead to secondary effects, such as lamellar-body formation and cellular injury. The chain of causality in this model runs opposite to that of the reigning paradigm, in which a primary lysosomal transport lesion results in impaired ER cholesterol metabolism [10,43,49]. Precision in cell cholesterol homeostasis could be enhanced by a two-tiered system of control - - the coarse adjustments made in the size of the regulatory ER pool by the upstream SSD-based sensor, NPC1, and the fine tuning achieved by the cholesterol-sensing elements in the ER. The latter proteins include ACAT, which apparently lacks an SSD, and the SSD-bearing proteins HMG-CoA reductase and SCAR Some forms of holoprosencephaly could arise from direct interactions between class 2 amphiphiles and the SSD on Patched. Alternatively, these malformations could be secondary to the aberrant sterol metabolism induced by these agents at a critical point in morphogenesis.
Acknowledgements We arc grateful to PA Beachy for providing unpublished findings and a copy of 155°° ] prior to its publication.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ° of outstanding interest 1.
Bloom M, Evans E, Mouritsen O: Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q Rev Biophys 1991, 24:293-397.
2.
Mouritsen OG, Jorgensen K: Dynamical order and disorder in lipid bilayers. Chem Phys Lipids 1994, 73:3-25.
3.
Hua X, Nohturfft A, Goldstein JL, Brown M: Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein. Ceil 1996, 87:415-426.
4. •,
Beachy PA, Cooper MK, Young KE, yon Kessler DP, Park W-J, Tanaka Hall TM, Leahy DJ, Porter J: Multiple roles of cholesterol in hedgehog protein biogenesis and signaling. Cold Spring Harb Syrup Quant Biol 1997, 62:191-204. Multiple connections are noted between cholesterol and early embryonic development. First is the tethering of a morphogenetic signal protein (Hedgehog) to the cell surface through a novel covalent bond to cholesterol. Secondly, an integral membrane protein (Patched), which is responsive to Hedgehog signals, has a putative sterol-sensing domain. Third, certain teratogens perturb cholesterol homeostasis. 5.
Lange Y, Echevarria F, Steck TL: Movement of zymosterol, a precursor of cholesterol, among three membranes in human fibroblasts. J Biol Chem 1991,266:21439-21443.
6. •e
Uittenbogaard A, Ying Y-s, Smart EJ: Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. Involvement in cholesterol trafficking. J Biol Chem 1998, 273:6525-6532. A fraction of caveolin is reported to form a soluble immunophilin-chaperone complex, which stimulates the movement of intracellular cholesterol to the plasma membrane. 7.
Lange Y, Strebel F, Steck TL: Role of the plasma membrane in cholesterol esterification in rat hepatoma cells. J Biol Chem 1993, 268:13838-13843.
8.
Brasaemle DL, Attie AD: Rapid intracellular transport of LDLderived cholesterol to the plasma membrane in cultured fibroblasts. J Lipid Res 1990, 31:103-112.
9.
Lange Y, Ye J, Steck TL: CirculaUon of cholesterol between lysosomes and the plasma membrane. J Bio/Chem 1998, 273:18915-18922.
10. Underwood KW, Jacobs NL, Howley A, Liscum L: Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane. J Bio/ Chem 1998, 273:4266-4274. 11. Lange Y, Ye J, Chin J: The fate of cholesterol exiting lysosomes. J Bio/Chem 1997, 272:17018-17022. 12. Freeman DA: Plasma membrane cholesterol: removal and insertion into the membrane and utilization as substrate for steroidogenesis. Endecrino/ogy 1989, 124:2527-2534. 13. Lange Y, Steck TL: Cholesterol homeostasis. Modulation by amphiphiles. J Bio/Chem 1994, 269:29371-29374. 14. Conrad PA, Smart El, Ying YS, Anderson RG, Bloom G: Caveolin cycles between plasma membrane caveolae and the Golgi complex bymicrotubule-dependent and microtubule-independent steps. J Ceil Bio/1995, 131:1421-1433. 15. Lange Y, Swaisgood MH, Ramos B, Steck TL: Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J Bio/Chem 1989, 264:3786-3793. 16. Liscum L, Underwood K: Intracellular cholesterol transport and compartmentation. J Bio/Chem 1995, 270:15443-15446. 17,
Harder T, Simons K: Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr Opin Ceil Bio/1997, 9:534-542.
18. Brown DA, London E: Structure of detergent-resistant membrane • domains: does phase separation occur in biological membranes? Biochem Biophys Res Commun 1997, 240:1-7. The significance of cholesteroFrich membrane bilayer domains that are stable in nonionic detergents is critically evaluated. 19. Scheiffele P, Verkade P, Fra AM, Virta H, Simons K, Ikonen E: • Caveolin-1 and -2 in the exocytic pathway of MDCK cells. J Ce// Biol 1998, 140:795-806. This paper provides evidence for both the vectorial movement of nascent caveolin isoforms and their role in targeting proteins to the surfaces of polarized epithelial cells. 20. Keller P, Simons K: Cholesterol is required for surface transport of he influenza virus hemaggliutinin. J Ceil Bio/1998, 140:137-1367. fact that cholesterol-sphingolipid rafts are vehicles in targeted exocytosis traffic is persuasively documented. 21. Fielding C J, Fielding PE: Intracellular cholesterol transport. J Lipid A Res 1997, 38:1503-1521. broad review, featuring the mechanisms that mediate cholesterol import from and export to high density lipoproteins. 22. Antonenkov V, Van Veldhoven P, Waelkens E, Mannaerts G: Substrate • specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyI-CoA thiolase purified from normal rat liver
Four cholesterol-sensing proteins Lange and Steck
peroxisomes. Sterol carrier protein 2/3-oxoacyI-CoA thiolase is
involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates. J Biol Chem 199?, 272:26023-26031. A strong contribution to the growing evidence suggesting that so-called 'sterol carrier proteins' may not serve this function in vivo. 23. Ikonen E: Molecular mechanisms of intracellular cholesterol transport. Curt Opin Lipido/1997, 8:60-64. 24. Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RG, Xu S, Krieger M: • Murine SR-BI, a high density lip•protein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J Bio/Chem 199?, 272:13242-13249. Caveolae are sterol-rich membrane domains that appear to carry out multiple structural and functional roles at the cell surface and in the cell interior. SR-BI, the receptor that mediates the transfer of cholesterol between plasma membranes and high-density plasma lip•proteins and therefore plays an important role in both cell and whole-body cholesterol homeostasis, is shown to be concentrated in caveolae. 25. Bist A, Fielding PE, Fielding C: Two sterel regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lip•protein free cholesterol. Proc Nat/ Acad Sci USA 1997, 94:10693-10698. Cholesterol is shown to positively regulate the expression of caveolin.
•
439
from in vivo competition studies. J Bio/ Chem 1998, 273:5765-5793. SREBPs are transcription factors that are activated by cleavage from the endoplasmic reticulum (ER) membrane under cholesterol control. The ER protein SCAP (SREBP cleavage-activating protein) is thought to regulate this proteolytic process, presumably using its sterol-sensing domain. This study provides evidence for the direct association of SCAP with its target, the SREBPs. 39. Scheek S, Brown MS, Goldstein J: Sphingomyelin depletion in • cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1. Proc Nat/Acad Sci USA 1997, 94:11179-11183.
The authors describe direct evidence that the proteolytic activation of SREBP is made in response to ER levels of cholesterol. 40. Hruban Z: Pulmonary and generalized lysosomal storage induced by amphiphilic drugs. Environ Health Perspect 1984, 55:53-76. 41. Schmitz G, Muller G: Structure and function of lamellar bodies, lipid-protein complexes involved in storage and secretion of cellular lipids. J Lipid Res 1991, 32:1539-1570. 42. McGookey DJ, Anderson R: Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells. J Cell Bio/1983, 97:1156-1166.
26. Hailstones D, Sleet LS, Parton RG, Stanley K: Regulation of caveolin • and caveolae by cholesterol in MDCK cells. J Lipid Res 1998, 39:369-379. Cholesterol is shown to positively regulate the expression of caveolin.
43. Buffer JD, Blanchette-Mackie J, Gotdin E, O'Neill RR, Carstea G, Roff CF, Patterson MC, Patei S, Cornly ME, Cooney A e t al.: Progesterone blocks cholesterol translocation from lysosomes. J Biol Chem 1992, 267:23797-23805.
27. Stocco D: A review of the characteristics of the protein required • for the acute regulation of steroid hormone biosynthesis: the case for the steroidogenic acute regulatory (STAR) protein. Proc Soc Exp Bio/ Med 1998, 217:123-129. An up-to-date overview of a newly discovered mitochondrial protein that regulates steroid•genesis.
44. MazzoneT, Krishna M, Lange Y: Progesterone blocks intracellular translocation of free cholesterol derived from cholesterol ester in macrophages. J Lipid Res 1995, 36:544-551.
28. Goldstein JL, Brown M: Regulation of the mevalonate pathway. Nature 1990, 343:425-430.
46. Metherall JE, Li H, Waugh K: Role of multidrug resistance Pglycoproteins in cholesterol biosynthesis. J Biol Chern 1996, 271:2634-2640.
29. Brown MS, Goldstein JL: The SREBP pathway: regulation of 00 cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Ceil 1997, 89:331-340. An overview of the system by which ER transcription factors regulate the expression of ER proteins that mediate cholesterol homeostasis. 30. Lange Y, Steck TL: The role of intracellular cholesterol transport in cholesterol homeostasis. Trends Ceil Bio/1996, 6:205-208. 31. Lange Y, Steck TL: Quantification of the pool of cholesterol • • associated with acyI-CoA cholesterol acyltransferase in human fibroblasts. J Bio/Chem 199?, 272:13103-13108. Most of the proteins that contol the level of cholesterol in cells are located in the endoplasmic reticulum (ER). The pool of cholesterol in the ER membrane is shown to be very small and very responsive to physiological stimuli. The size of the ER cholesterol compartment therefore appears to be regulated so as to serve as a critical element in cholesterol homeostasis. 32. Cheng D, Chang CC, Clu X, Chang T: Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system. J Bio/Chem 1995, 270:685-695. 33. Chang TY, Chang CC, Cheng D: Acyl-coenzyme A:cholesterol acyltransferase. Annu Rev Biochem 1997, 66:613-638. A definitive review of a critical element in cholesterol homeostasis. 34. •lender EH, Simoni R: The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase. J Bio/ Chem 1992, 267:4223-4235. 35. McGee TP, Cheng HH, Kumagai H, Ornura S, Sirnoni R: Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis. J Bio/Chem 1996, 271:25630-25638. 36. Sakai J, Duncan EA, Rawson RB, Hua X, Brown MS, Goldstein J: Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment. Ceil 1996, 85:1037-1046. 37. Sakai J, Nohturfft A, Cheng D HY, Brown MS, Goldstein J: Identification of complexes between the CO•H-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein. J Bio/Chem 1997, 272:20213-20221. 38. Sakai J, Nohturfft A, Goldstein JL, Brown MS: Cleavage of sterol • regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence
45. Metherall JE, Waugh K, Li H: Progesterone inhibits cholesterol biosynthesis in cultured cells. Accumulation of cholesterol precursors. J Biol Chem 1996, 271:2627-2633.
4?.
FieldFJ, Born E, Murthy S, Mathur S: Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in CaCo-2 cells and differs from the transport of
plasma membrane cholesterol to the endoplasmic reticulum. J Lipid Res 1998, 39:333-343. 48. Debry P, Nash EA, Neklason DW, Metherall J: Role of multidrug resistance P-glycoproteins in cholesterol esterification. J Biol Chem 1997, 272:1026-1031. 49. Pentchev PG, Vanier MT, Suzuki K, Patterson MC: Niemann-Pick disease type C: a cellular cholesterol lipid•sis. In The Metabofic and Molecular Bases of Inherited Disease. Edited by Scriver CR, Beaudet AL, Sly WS, Valle D, Stanbury JB, Wyngaarden JB, Fredrickson DS. New York: McGraw-Hill; 1995:2625-2639. 50. Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings 00 C, Gu J, Rosenfeld MA, Pavan WJ, Krizman DB et aL: Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 199"7, 277:228-231. The identification of NPC1, the gene for a fatal cholesterol storage disease, suggests that it is an integral membrane glycoprotein with a sterol-sensing domain. 51. Loftus SK, Morris JA, Carstea ED, Gu JZ, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld MA, Tagle DA et al.: Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 199?, 277:232-235. 52. Pentchev PG, Brady RO, Blanchette-Mackie El, Vanier MT, Carstea ED, Parker CC, Goldin E, Roff CF: The Niemann-Pick C lesion and its relationship to the intracellular distribution and utilization of LDL cholesterol. Biochim Biophys Acta 1994, 1225:235-243. 53. Herz J, Willnow TE, Farese RJ: Cholesterol, hedgehog and embryogenesis. Nat Genet 199?, 15:123-124. 54. Porter JA, Young KE, Beachy P: Cholesterol modification of hedgehog signaling proteins in animal development. Science 1996, 274:255-259. 55. Cooper MK, Porter JA, Young KE, Beachy P: Teratogen-mediated 00 inhibition of target tissue response in Sonic hedgehog signaling. Science 1998, 280:1603-1607. Amphiphiles that perturb sterol homeostasis affect the morphogenetic control system, which responds to Hedgehog. The target of these amphiphiles may be Patched, a sterol-sensing receptor for Hedgehog.