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Cellular sterol trafficking and metabolism: spotlight on structure
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Cellular sterol trafficking and metabolism: spotlight on structure Elina Ikonen and Maurice Jansen Cholesterol is the main but not the only sterol in cell membranes of higher eukaryotes. Currently, there is an increasing interest toward structurally different sterols, because their membrane partitioning, trafficking, and metabolic properties may differ considerably from those of cholesterol. There is also growing information on specific sterol–protein interactions and their functional consequences, as exemplified by NPC proteins and select ABC-transporters. Several aspects of sterol trafficking and homeostasis are conserved between eukaryotes, and novel, unanticipated findings in this area have recently been made, particularly from genetic screens in yeast. This includes a novel, reversible modification of the sterol structure that affects the choice of transport route. Address Institute of Biomedicine/Anatomy, Haartmaninkatu 8, 00014 University of Helsinki, Finland Corresponding author: Ikonen, Elina ([email protected])
Current Opinion in Cell Biology 2008, 20:371–377 This review comes from a themed issue on Membranes and organelles Edited by Grac¸a Raposo and Harald Stenmark Available online 24th May 2008 0955-0674/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2008.03.017
Introduction In cell biology, cholesterol is often regarded as a longlived structural constituent of membranes and the only important membrane sterol in mammalian cells. This view is rapidly giving way to a more dynamic view on the role of sterols in eukaryotes, which takes into account their structural heterogeneity and differential behavior in membranes. This notion on the importance of sterol structural specificity in cell biology is somewhat reminiscent to the earlier recognition of phosphoinositide phosphates (PIPs) with different molecular species and specific functional roles. Derivatives of a major membrane lipid phosphatidylinositol represent minor, but functionally ‘active’ lipid species. Analogously, many of the quantitatively minor cholesterol related sterols may display specific effects. Research on phosphoinositides has been spurred by the identification of specific protein modules that bind individual PIPs. For sterols, the picture appears more complex, partly because information on sterol-binding areas may be difficult to deduce from www.sciencedirect.com
the protein primary structure or from in vitro-binding studies, and membrane protein structures with sterols are not trivial to solve. Nevertheless, important steps have been taken forward in this area. Many of the key proteins involved in sterol trafficking have traditionally been discovered by identifying gene defects that underlie human disorders of cholesterol trafficking [1]. The availability of genome sequences from an increasing number of species and the possibility to efficiently conduct genome wide screens, also for lipid trafficking, is now changing this scenario. These strategies are providing entirely new links between sterol and other metabolic pathways. The challenge here is that this information needs to be integrated into the existing knowledge on sterol trafficking and metabolism. In this review, we will focus on selected aspects of cellular sterol trafficking, where progress has recently been made. This encompasses lessons from yeast genetic screens, structural considerations on sterol modifications and protein–sterol interactions and finally, examples of human disease aspects related to sterol specificity. The sterol structures discussed are indicated in Figure 1, and the cellular sterol trafficking pathways and sterol-binding proteins introduced are schematically illustrated in Figure 2. We would like to forward the reader to recent reviews on other interesting aspects of cellular sterol trafficking [2,3,4] and on more general discussion on intracellular cholesterol trafficking routes [5].
New insights into sterol trafficking from yeast genetic screens Saccharomyces cerevisiae does not take up sterols in the presence of oxygen (but rather synthesizes ergosterol, the major membrane sterol, and the other sterols needed). However, it becomes dependent on exogenously supplied sterols in anaerobic conditions, as sterol synthesis requires oxygen. In two recent studies, genome-wide screens were used to identify proteins involved in sterol uptake in yeast [6,7]. In one, sterol uptake assays were carried out in mutants lacking heme, thus mimicking anaerobic conditions [6]. In the other, drug-induced disruptions in sterol homeostasis (nystatin, lovastatin) were employed [7]. One large class of the sterol uptake mutants identified using heme-deficient cells affect the expression and localization of the ABC transporters Aus1p and Pdr11p, already known to be required for sterol uptake under unaerobic conditions [8,9]. Another large class of mutants isolated is affected in mitochondrial functions. Several of them have mutations in the soluble F1 sector of the ATPCurrent Opinion in Cell Biology 2008, 20:371–377
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Figure 1
linked and what specific mitochondrial function(s) is required for sterol uptake under anaerobic conditions await to be elaborated. The other screen produced information on the relationships of sterol storage and transport, and identified three genes potentially governing sterol trafficking, namely ARV1, PLC1 and PTC1 [7]. In plc1D and ptc1D mutants, unesterified sterol is redistributed to endosomes, by yet unknown mechanisms. The arv1D mutant has previously been shown to accumulate sterols in intracellular membranes [10,11]. Moreover, recent results indicate that Arv1p is required for the efficient delivery of an early glycosylphosphatidylinositol (GPI) anchor intermediate to the ER lumen as well as regulation of ceramide transport from the ER and maintenance of intracellular sterol amounts and distribution [12]. Unesterified sterol accumulates in the ER and lipid particles but how this is connected to the other phenotypes, remains to be explained. Possibly, GPI anchor synthesis regulates vesicular ceramide transport from the ER because of the coexport of GPI-anchored proteins and ceramides, and sterol then becomes sequestered because of its sphingolipid affinity [13].
Sterol modifications and significance of structural specificity Sterol precursors and oxysterols
Structures of the sterol metabolites are discussed in this review. The numbering of carbon atoms in the steroid backbone and the stereo configuration are indicated for cholesterol. Ergosterol, the predominant sterol in yeast, is shown next to cholesterol. Cholesterol biosynthetic precursors are illustrated on the left, oxysterols on the right and sterol esters at the bottom. The structural differences compared to cholesterol are highlighted with color: red, methyl group; yellow, double/single bond; blue, keto- or hydroxyl-group.
synthase or a chaperone required for its assembly. Although a defect in maintaining the electrochemical gradient across the mitochondrial inner membrane would explain viability defects, it may not readily explain the sterol uptake defect. Interestingly, several of the sterol uptake mutants as well as an aus1Dpdr1D mutant have electron-dense mitochondrial inclusions, suggesting that the aberrant mitochondria may result from impaired sterol uptake [6]. How these findings are mechanistically Current Opinion in Cell Biology 2008, 20:371–377
Cholesterol biosynthesis proceeds via multiple sterol intermediates (with 19 enzymatic steps from the first sterol, lanosterol, to cholesterol) [14,15]. Thus, whenever cells synthesize cholesterol (or ergosterol in yeast), these precursor sterols are also present. In many cases, they represent minor proportions of the total sterols because of their rapid conversion to the next intermediate and/or release from cells [16–18]. However, in specific cell types, such as developing astrocytes select precursor sterols may represent up to half of total sterols [19]. This may be relevant for regulating protein functions that depend on ordered membrane domains [20,21]. For instance, desmosterol exhibits a weaker interaction with caveolin than cholesterol and perturbs the structure of caveolae [20]. Although the rate-limiting enzyme of cholesterol biosynthesis, hydroxyglutaryl-CoA reductase, has been intensively investigated (e.g. recently regarding its inactivation [22,23]) regulation of post-lanosterol enzymes is less well understood. Lanosterol demethylase belongs to P450 enzymes, monooxygenases that are involved in many key physiological processes and subject to regulation by hemoproteins (as elucidated e.g. in [24]). There is also new information on the regulation of a non-P450 sterol biosynthetic enzyme, dehydrocholesterol 24reductase (DHCR24, see below). Furthermore, sterols are modified by oxidation (either enzymatic or auto-oxidation) to produce a variety of oxysterols that are more hydrophilic than the parental www.sciencedirect.com
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Figure 2
Schematic illustration of the cellular sterol trafficking pathways and sterol-binding proteins is discussed in this review. Sterol transport is indicated by thick arrows and sterol-binding by specific proteins by thin arrows. Cholesterol precursors are shown in dark green (PreC, several cholesterol precursors; Desmo, desmosterol), oxysterols in light green (OH-C, several oxysterols; 25-HC, 25-hydroxycholesterol; 7-KC, 7-ketocholesterol), cholesterol (Chol) in light blue and cholesteryl acetate (CAc) in dark blue. MACPF, membrane attack complex/perforin; ABCG1 and ABCG4, ATPbinding cassette transporters G1 and G4; NPC1 and NPC2, Niemann-Pick type C proteins 1 and 2; INSIG, insulin-induced gene; SCAP, sterol response element-binding protein cleavage activating protein; PM, plasma membrane; LE, late endosome; ER, endoplasmic reticulum. Numbers in brackets indicate the references, in which the findings on sterol trafficking/binding are reported.
compounds because of additional hydroxy- or ketogroups. Analogously to the sterol precursors, oxysterols associate with membranes and may display differential distributions between cell types. Members of the oxysterol-binding protein (OSBP) family (ORPs, OSBPrelated proteins) show distinct, though partially overlapping binding-specificities for different sterols, and evidence from yeast suggests a function for these proteins in the nonvesicular intracellular transport of sterols [25]. However, for mammalian ORPs functional studies point to more diverse roles and rather suggest that these proteins mainly act as sterol sensors [26]. At present, little is known of the trafficking of sterol precursors or oxysterols in higher eukaryotes. Lipid rafts were suggested to participate in the retrograde movement of sterol precursors from the plasma membrane to the ER as raft disruption (by cholesterol depletion) retards the conversion of precursors to cholesterol [27]. Another interpretation might be that cholesterol removal does not perturb the transport process per se but increases the affinity of sterol precursors to the plasma membrane.
active site in the ER lumen. Sterol acetylation acts as a detoxification mechanism, resulting in the secretion of the acetylated sterol via the secretory pathway. Substrate specificity of the deacetylase Say1p appears to control, which sterols are deacetylated and retained in the cell. The steroid hormone precursor pregnenolone is also acetylated but its export is independent of secretion, indicating that yeast has several mechanisms to export acetylated sterols. Importantly, sterol deacetylation is conserved in metazoans, with the human arylacetamide deacetylase capable of providing deacetylation activity when expressed in yeast [28]. In vertebrates, glucuronidation and sulfonation are known modifications for detoxification and excretion of sterols and steroids [29,30] but whether acetylation could also operate as such, remains open. Mechanistically, an interesting question is also how cholesterol acetate — that is even more hydrophobic than cholesterol — is rendered soluble in the ER lumen or in the extracellular milieu.
Novel modifications
It is evident that structural information on the interactions between sterols and proteins is crucial for understanding protein mediated sterol transport and the mechanisms by which sterols affect proteins. Also in this area, several new findings have been made.
A novel, reversible sterol modification was recently identified in S. cerevisiae, the sterol acetylation/deacetylation cycle [28]. Both the acetyltransferase and deacetylase enzymes are ER-membrane proteins, with a putative www.sciencedirect.com
Structural considerations on protein–sterol interactions
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Membrane attack complex proteins
Papers published in the same issue of Science determined the crystal structures of two membrane attack complex/ perforin (MACPF) domains and revealed a novel role for cholesterol in vertebrate immunity [31,32]. The structure of MACPF appears similar to that of the pore-forming cholesterol-dependent cytolysins (CDCs), a large family of bacterial toxins capable of generating oligomeric pores in membranes by specifically binding to cholesterol. The authors propose that MACPF proteins function in a CDC like manner. Interestingly, besides immunity MACPF proteins also play important roles in embryonic development and neuronal cell migration [33,34]. It therefore appears plausible that cholesterol is involved in regulating MACPF dependent aspects of these processes. SREBP-machinery
Recent studies by Brown and Goldstein have gained insight into the roles of cholesterol and oxysterols in regulating the transport of sterol regulatory elementbinding proteins (SREBPs) from the ER. It has been known for some time that both cholesterol and 25-hydroxycholesterol (25-HC) induce the interaction between Insig and Scap and thereby retain SREBP in the ER [35]. This prevents SREBP from being transported to and processed in the Golgi and consequently inhibits the expression of genes involved in sterol biosynthesis. It now appears that cholesterol and 25-HC regulate this pathway by binding different proteins. Cholesterol binds Scap causing Scap to bind Insig, whereas 25-HC binds Insig, causing Insig to bind Scap [36]. Structural analysis suggests that Scap-Insig binding renders the hexapeptide sorting signal (MELADL) in Scap inaccessible to COPII proteins [37], possibly by altering its location with respect to the membrane.
argue that this might be caused by the presence of other sterol binding sites (NPC1 contains a sterol-sensing domain outside loop-1) or by some nonphysiological aspects of the in vitro binding assay. In contrast to NPC1, NPC2 is a soluble late endosomal protein capable of transporting cholesterol in vitro. New structural studies demonstrate that NPC2 can bind a wide array of sterols besides cholesterol. These include cholesterol precursors, plant sterols and oxysterols [40]. This apparent promiscuity might be the consequence of a malleable binding site, as supported by the observation that apoNPC2 contains a loosely packed hydrophobic core, too small to accommodate a sterol [41]. In the presence of sterol, the pocket expands by structural alterations, including the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function [41]. Notably, the list of ligands did not include various glycolipids, phospholipids or fatty acids, suggesting a specific role for NPC2 in sterol transport [40].
Disease aspects related to sterol specificity Several recent studies highlight the importance of sterol structure in the pathogenesis of human diseases, or the respective mouse models. Mouse models have been generated for several inborn errors of cholesterol biosynthesis, such as Smith-Lemli-Opitz syndrome (caused by defective 7-dehydrocholesterol reductase, DHCR7) and lathosterolosis (caused by defective lathosterol-5-desaturase, SC5D). Recent studies in these mice revealed that the accumulating 7-dehydrocholesterol and lathosterol cause secretory granule malformation, apparently because of decreased membrane curvature [42]. The role of specific sterols has also been investigated in relation to Alzheimer’s disease, atherosclerosis and Huntington’s disease, as discussed below.
NPC proteins
DHCR24/seladin-1
NPC proteins are necessary for the exit of cholesterol from the endocytic system and their deficiency results in Niemann-Pick type C (NPC) disease. Although many groups have studied these proteins, their precise mechanisms of action in cholesterol transport remain unknown. Recent structural studies on both NPC1 and NPC2 have revealed some surprising features. NPC1 is a late endosomal membrane protein containing 13 putative transmembrane helices. Unexpectedly, this protein was encountered in a search for a membrane protein that binds 25-HC [38]. The binding site was localized to luminal loop-1, a 240 amino acid domain which shows strong sequence conservation in vertebrates and yeast [39]. Besides oxysterols (25-HC, 27-HC, 24(S)-HC) this site was shown to bind cholesterol, albeit less avidly than 25-HC [38]. Surprisingly, a point mutant that abolished binding of 25-HC and cholesterol was nevertheless able to revert the NPC phenotype (the accumulation of cholesterol in late endocytic organelles) [39]. The authors
DHCR24 is an oxidoreductase that catalyzes desmosterol-cholesterol conversion. Interestingly, the same protein has also been described in a different context: to be downregulated in the affected areas of Alzheimer’s disease brain (therefore named seladin-1 for selective Alzheimer’s disease indicator-1 [43]) and to confer resistance against oxidative stress and apoptotic cell death [43– 45]. Until recently, it has been difficult to reconcile the cholesterol biosynthetic and anti-apoptotic roles of the protein. However, new observations by Kuehnle et al. [46] clarify a number of issues. They show that DHCR24/seladin-1 is upregulated in an acute response and downregulated in a chronic response to oxidative stress and that the protective effects against apoptosis are observed in both cases. The acute protective effect seems to be related to cholesterol production and the fact that cholesterol offers better protection against oxidative apoptotic insult than desmosterol (possibly by counteracting lipid peroxidation and maintaining membrane
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integrity). By contrast, the prosurvival impact of low DHCR24/seladin-1 levels appears to be mediated by the tumor suppressor protein p53 in a manner that is independent of DHCR24 activity, as also reported earlier [47], and thus unrelated to membrane cholesterol concentration. Seladin-1 binding to p53 is known to result in p53 accumulation via its protection from proteasomal degradation [47]. Conversely, lowered seladin-1 levels provide deficient stabilization of p53 and therefore protect cells from p53 mediated apoptotic death [46]. Interestingly, the protective effect of DHCR24/seladin1 in Alzheimer’s disease has been linked to its role in as DHCR24 in cholesterol synthesis, via increased recruitment of cholesterol and detergent-resistant membrane (DRM) proteins into rafts and consequent reduction in beta-amyloid generation [48]. Whether this might also involve the seladin-1 function of the protein is an open question.
reporting similar observations in another HD mouse model and more remarkably, show that mice overexpressing wild-type huntingtin display higher activity of the cholesterol biosynthetic pathway and also higher levels of brain and plasma cholesterol, cholesterol precursors and 24(S)-HC with respect to wild-type animals. This suggests that normal huntingtin may play a role in cholesterol biosynthesis [54]. Intriguingly, in another study cholesterol was found to accumulate in brains and cultured neurons expressing mutant huntingtin [55]. This was linked to caveolin-1 interaction and inhibition of clathrin-independent (potentially caveolar) endocytosis by mutant huntingtin. How these findings can be reconciled with the results above is not clear, but perhaps the different mouse models and time-points of analysis contribute. In any case, both data sets agree with the conclusion that brain sterol homeostasis is perturbed in HD.
ABC-transporters
For the past years, there has been increasing appreciation on the critical role of ABC-transporters in cholesterol trafficking, particularly as proteins functioning in the ATP-dependent release of cholesterol from cells to high-density lipoproteins (HDL) or its major apoprotein ApoA-I [49]. Recent reports reveal not only that these proteins are involved in the efflux of sterol precursors and oxysterols, but also that individual transporters exhibit specificity for select sterols. The efflux of 7-ketocholesterol, the major oxysterol of oxidized low-density lipoproteins (oxLDL) and atherosclerotic lesions, is entirely dependent on the ABCG1 protein in macrophages [50]. Surprisingly, the ABCA1 transporter that is critical for cholesterol efflux to ApoA-I, is not involved. Accordingly, protection of macrophages from oxLDL-induced apoptosis specifically requires ABCG1 and reflects its ability to promote oxysterol efflux from macrophages. In astrocytes, on the contrary, the active transport of desmosterol to HDL is dependent on the overlapping functions of ABCG1 and ABCG4 transporters [51]. Huntingtin
In the central nervous system, cholesterol is synthesized locally and eliminated by conversion to an oxysterol, 24(S)-HC, that crosses the blood–brain barrier [52]. There seems to be an increasing number of neurodegenerative diseases, in which the brain sterol balance is implicated. An example is Huntington’s disease (HD) caused by an expanded trinucleotide repeat in the huntingtin gene. Cholesterol biosynthetic pathway was shown to be altered in fibroblasts and brains of HD patients as well as in a HD mouse model [53]. This seems to be attributable to a mutant huntingtin-dependent decrease in the translocation of the active SREBP to the nucleus [53]. Recent data strengthen the case by www.sciencedirect.com
Conclusions and perspectives It is becoming increasingly evident that besides their main structural membrane sterol, eukaryotic cells use a variety of minor sterol metabolites to generate specificity to sterol trafficking and protein–sterol interactions. Clearly, this characteristic is not limited to cell types that are traditionally considered as sterol processing, such as steroidogenic cells — that produce steroid hormones from cholesterol — or hepatocytes — that use cholesterol to generate bile acids. Several sterol-interacting proteins have broader specificities for sterol binding than initially anticipated, at least in in vitro settings. In living cells where kinetic and steric considerations generate more restrictions, the physiologically relevant interactions are probably more limited. The growing awareness of a multitude of sterol species in cells has important implications. It highlights the relevance of not only resolving them but also of establishing new tools to follow their cellular itineraries.
Acknowledgements The authors would like to thank Matts Linder and Aino Mutka for discussions. Our work was supported by the Academy of Finland, Sigrid Juselius Foundation, Finnish Heart Foundation and Helsinki Biomedical Graduate School.
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36. Radhakrishnan A, Ikeda Y, Kwon HJ, Brown MS, Goldstein JL: Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig. Proc Natl Acad Sci U S A 2007, 104:6511-6518. This study defines Insig as a membrane bound oxysterol-binding protein with specificity for cholesterol derivatives that possess hydroxyl groups on the side chain.
46. Kuehnle K, Crameri A, Kalin RE, Luciani P, Benvenuti S, Peri A, Ratti F, Rodolfo M, Kulic L, Heppner FL et al.: Prosurvival effect of DHCR24/Seladin-1 in acute and chronic responses to oxidative stress. Mol Cell Biol 2008, 28:539-550. This study solved the paradox that DHCR24 can both confer senescence and protection against oxidative stress by showing that that it functions by two mechanistically independent pathways; one involves cholesterol and the other p53.
37. Sun LP, Seemann J, Goldstein JL, Brown MS: Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins. Proc Natl Acad Sci U S A 2007, 104: 6519-6526. This article shows that both cholesterol binding to Scap and oxysterol binding to Insig independently induce a similar conformational change in the cytoplasmic loop 6 of Scap that prevents the COPII proteins from gaining access to the MELADL sorting sequence. The authors hypothesize that the distance of the MELADL sequence from the membrane is crucial for sterol-mediated regulation of COPII accessibility of Scap. 38. Infante RE, Abi-Mosleh L, Radhakrishnan A, Dale JD, Brown MS, Goldstein JL: Purified NPC1 protein. I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein. J Biol Chem 2008, 283:1052-1063. This study identified NPC1 as an oxysterol-binding protein but showed that NPC1 is not required for the known regulatory actions of oxysterols. 39. Infante RE, Radhakrishnan A, Abi-Mosleh L, Kinch LN, Wang ML, Grishin NV, Goldstein JL, Brown MS: Purified NPC1 protein. II. Localization of sterol binding to a 240-amino acid soluble luminal loop. J Biol Chem 2008, 283:1064-1075. In this article NPC1 was shown to contain a sterol-binding site in a soluble luminal loop. The authors propose that this binding site is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities. 40. Liou HL, Dixit SS, Xu S, Tint GS, Stock AM, Lobel P: NPC2, the protein deficient in Niemann-Pick C2 disease, consists of multiple glycoforms that bind a variety of sterols. J Biol Chem 2006, 281:36710-36723. 41. Xu S, Benoff B, Liou HL, Lobel P, Stock AM: Structural basis of sterol binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 disease. J Biol Chem 2007, 282:23525-23531. By analyzing the structure of both apo-bound and sterol-bound NPC2, this study provides evidence for a malleable-binding site which is consistent with the previously documented broad range of sterol ligand specificity. 42. Gondre-Lewis MC, Petrache HI, Wassif CA, Harries D, Parsegian A, Porter FD, Loh YP: Abnormal sterols in cholesterol-deficiency diseases cause secretory granule malformation and decreased membrane curvature. J Cell Sci 2006, 119:1876-1885. By using cells with a defect in cholesterol biosynthesis this study shows that sterol structure is important for granule formation and correlates granule size and bending rigidity to sterol structure. 43. Greeve I, Hermans-Borgmeyer I, Brellinger C, Kasper D, GomezIsla T, Behl C, Levkau B, Nitsch RM: The human DIMINUTO/ DWARF1 homolog seladin-1 confers resistance to Alzheimer’s disease-associated neurodegeneration and oxidative stress. J Neurosci 2000, 20:7345-7352. 44. Luciani P, Gelmini S, Ferrante E, Lania A, Benvenuti S, Baglioni S, Mantovani G, Cellai I, Ammannati F, Spada A et al.: Expression of the antiapoptotic gene seladin-1 and octreotide-induced apoptosis in growth hormone-secreting and nonfunctioning pituitary adenomas. J Clin Endocrinol Metab 2005, 90:6156-6161.
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47. Wu C, Miloslavskaya I, Demontis S, Maestro R, Galaktionov K: Regulation of cellular response to oncogenic and oxidative stress by Seladin-1. Nature 2004, 432:640-645. 48. Crameri A, Biondi E, Kuehnle K, Lutjohann D, Thelen KM, Perga S, Dotti CG, Nitsch RM, Ledesma MD, Mohajeri MH: The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Abeta generation in vivo. EMBO J 2006, 25:432-443. This paper links the protective effect of DHCR24/Seladin-1 against Abmediated toxicity with its sterol reductase activity by showing that lack of DHCR24 reduces g-secretase DRM localization. 49. Brewer HB Jr: HDL metabolism and the role of HDL in the treatment of high-risk patients with cardiovascular disease. Curr Cardiol Rep 2007, 9:486-492. 50. Terasaka N, Wang N, Yvan-Charvet L, Tall AR: High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7ketocholesterol via ABCG1. Proc Natl Acad Sci U S A 2007, 104:15093-15098. This study indicates a specific role for ABCG1 in promoting efflux of 7ketocholesterol and related oxysterols from macrophages onto HDL and in protecting these cells from oxysterol-induced cytotoxicity. 51. Wang N, Yvan-Charvet L, Lutjohann D, Mulder M, Vanmierlo T, Kim TW, Tall AR: ATP-binding cassette transporters G1 and G4 mediate cholesterol and desmosterol efflux to HDL and regulate sterol accumulation in the brain. FASEB J 2008, 22:1073-1082. This article provides the first direct evidence for a role of Abcg4 in efflux of sterol from astrocytes to HDL in the brain. The active efflux of desmosterol was shown to involve Abcg1, Abcg4, and Abca1, suggesting that this sterol has an important role in brain sterol homeostasis. 52. Dietschy JM, Turley SD: Thematic review series: brain Lipids. Cholesterol metabolism in the central nervous system during early development and in the mature animal. J Lipid Res 2004, 45:1375-1397. 53. Valenza M, Rigamonti D, Goffredo D, Zuccato C, Fenu S, Jamot L, Strand A, Tarditi A, Woodman B, Racchi M et al.: Dysfunction of the cholesterol biosynthetic pathway in Huntington’s disease. J Neurosci 2005, 25:9932-9939. 54. Valenza M, Carroll JB, Leoni V, Bertram LN, Bjorkhem I, Singaraja RR, Di Donato S, Lutjohann D, Hayden MR, Cattaneo E: Cholesterol biosynthesis pathway is disturbed in YAC128 mice and is modulated by huntingtin mutation. Hum Mol Genet 2007, 16:2187-2198. This study suggests that huntingtin influences cholesterol biosynthesis by showing that overexpression of mutant or wild-type huntingtin in mice results in the upregulation or downregulation of cholesterol biosynthesis, respectively. 55. Trushina E, Singh RD, Dyer RB, Cao S, Shah VH, Parton RG, Pagano RE, McMurray CT: Mutant huntingtin inhibits clathrinindependent endocytosis and causes accumulation of cholesterol in vitro and in vivo. Hum Mol Genet 2006, 15:3578-3591.
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Cellular sterol trafficking and metabolism: spotlight on structure Elina Ikonen and Maurice Jansen Cholesterol is the main but not the only sterol in cell membranes of higher eukaryotes. Currently, there is an increasing interest toward structurally different sterols, because their membrane partitioning, trafficking, and metabolic properties may differ considerably from those of cholesterol. There is also growing information on specific sterol–protein interactions and their functional consequences, as exemplified by NPC proteins and select ABC-transporters. Several aspects of sterol trafficking and homeostasis are conserved between eukaryotes, and novel, unanticipated findings in this area have recently been made, particularly from genetic screens in yeast. This includes a novel, reversible modification of the sterol structure that affects the choice of transport route. Address Institute of Biomedicine/Anatomy, Haartmaninkatu 8, 00014 University of Helsinki, Finland Corresponding author: Ikonen, Elina ([email protected])
Current Opinion in Cell Biology 2008, 20:371–377 This review comes from a themed issue on Membranes and organelles Edited by Grac¸a Raposo and Harald Stenmark Available online 24th May 2008 0955-0674/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2008.03.017
Introduction In cell biology, cholesterol is often regarded as a longlived structural constituent of membranes and the only important membrane sterol in mammalian cells. This view is rapidly giving way to a more dynamic view on the role of sterols in eukaryotes, which takes into account their structural heterogeneity and differential behavior in membranes. This notion on the importance of sterol structural specificity in cell biology is somewhat reminiscent to the earlier recognition of phosphoinositide phosphates (PIPs) with different molecular species and specific functional roles. Derivatives of a major membrane lipid phosphatidylinositol represent minor, but functionally ‘active’ lipid species. Analogously, many of the quantitatively minor cholesterol related sterols may display specific effects. Research on phosphoinositides has been spurred by the identification of specific protein modules that bind individual PIPs. For sterols, the picture appears more complex, partly because information on sterol-binding areas may be difficult to deduce from www.sciencedirect.com
the protein primary structure or from in vitro-binding studies, and membrane protein structures with sterols are not trivial to solve. Nevertheless, important steps have been taken forward in this area. Many of the key proteins involved in sterol trafficking have traditionally been discovered by identifying gene defects that underlie human disorders of cholesterol trafficking [1]. The availability of genome sequences from an increasing number of species and the possibility to efficiently conduct genome wide screens, also for lipid trafficking, is now changing this scenario. These strategies are providing entirely new links between sterol and other metabolic pathways. The challenge here is that this information needs to be integrated into the existing knowledge on sterol trafficking and metabolism. In this review, we will focus on selected aspects of cellular sterol trafficking, where progress has recently been made. This encompasses lessons from yeast genetic screens, structural considerations on sterol modifications and protein–sterol interactions and finally, examples of human disease aspects related to sterol specificity. The sterol structures discussed are indicated in Figure 1, and the cellular sterol trafficking pathways and sterol-binding proteins introduced are schematically illustrated in Figure 2. We would like to forward the reader to recent reviews on other interesting aspects of cellular sterol trafficking [2,3,4] and on more general discussion on intracellular cholesterol trafficking routes [5].
New insights into sterol trafficking from yeast genetic screens Saccharomyces cerevisiae does not take up sterols in the presence of oxygen (but rather synthesizes ergosterol, the major membrane sterol, and the other sterols needed). However, it becomes dependent on exogenously supplied sterols in anaerobic conditions, as sterol synthesis requires oxygen. In two recent studies, genome-wide screens were used to identify proteins involved in sterol uptake in yeast [6,7]. In one, sterol uptake assays were carried out in mutants lacking heme, thus mimicking anaerobic conditions [6]. In the other, drug-induced disruptions in sterol homeostasis (nystatin, lovastatin) were employed [7]. One large class of the sterol uptake mutants identified using heme-deficient cells affect the expression and localization of the ABC transporters Aus1p and Pdr11p, already known to be required for sterol uptake under unaerobic conditions [8,9]. Another large class of mutants isolated is affected in mitochondrial functions. Several of them have mutations in the soluble F1 sector of the ATPCurrent Opinion in Cell Biology 2008, 20:371–377
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Figure 1
linked and what specific mitochondrial function(s) is required for sterol uptake under anaerobic conditions await to be elaborated. The other screen produced information on the relationships of sterol storage and transport, and identified three genes potentially governing sterol trafficking, namely ARV1, PLC1 and PTC1 [7]. In plc1D and ptc1D mutants, unesterified sterol is redistributed to endosomes, by yet unknown mechanisms. The arv1D mutant has previously been shown to accumulate sterols in intracellular membranes [10,11]. Moreover, recent results indicate that Arv1p is required for the efficient delivery of an early glycosylphosphatidylinositol (GPI) anchor intermediate to the ER lumen as well as regulation of ceramide transport from the ER and maintenance of intracellular sterol amounts and distribution [12]. Unesterified sterol accumulates in the ER and lipid particles but how this is connected to the other phenotypes, remains to be explained. Possibly, GPI anchor synthesis regulates vesicular ceramide transport from the ER because of the coexport of GPI-anchored proteins and ceramides, and sterol then becomes sequestered because of its sphingolipid affinity [13].
Sterol modifications and significance of structural specificity Sterol precursors and oxysterols
Structures of the sterol metabolites are discussed in this review. The numbering of carbon atoms in the steroid backbone and the stereo configuration are indicated for cholesterol. Ergosterol, the predominant sterol in yeast, is shown next to cholesterol. Cholesterol biosynthetic precursors are illustrated on the left, oxysterols on the right and sterol esters at the bottom. The structural differences compared to cholesterol are highlighted with color: red, methyl group; yellow, double/single bond; blue, keto- or hydroxyl-group.
synthase or a chaperone required for its assembly. Although a defect in maintaining the electrochemical gradient across the mitochondrial inner membrane would explain viability defects, it may not readily explain the sterol uptake defect. Interestingly, several of the sterol uptake mutants as well as an aus1Dpdr1D mutant have electron-dense mitochondrial inclusions, suggesting that the aberrant mitochondria may result from impaired sterol uptake [6]. How these findings are mechanistically Current Opinion in Cell Biology 2008, 20:371–377
Cholesterol biosynthesis proceeds via multiple sterol intermediates (with 19 enzymatic steps from the first sterol, lanosterol, to cholesterol) [14,15]. Thus, whenever cells synthesize cholesterol (or ergosterol in yeast), these precursor sterols are also present. In many cases, they represent minor proportions of the total sterols because of their rapid conversion to the next intermediate and/or release from cells [16–18]. However, in specific cell types, such as developing astrocytes select precursor sterols may represent up to half of total sterols [19]. This may be relevant for regulating protein functions that depend on ordered membrane domains [20,21]. For instance, desmosterol exhibits a weaker interaction with caveolin than cholesterol and perturbs the structure of caveolae [20]. Although the rate-limiting enzyme of cholesterol biosynthesis, hydroxyglutaryl-CoA reductase, has been intensively investigated (e.g. recently regarding its inactivation [22,23]) regulation of post-lanosterol enzymes is less well understood. Lanosterol demethylase belongs to P450 enzymes, monooxygenases that are involved in many key physiological processes and subject to regulation by hemoproteins (as elucidated e.g. in [24]). There is also new information on the regulation of a non-P450 sterol biosynthetic enzyme, dehydrocholesterol 24reductase (DHCR24, see below). Furthermore, sterols are modified by oxidation (either enzymatic or auto-oxidation) to produce a variety of oxysterols that are more hydrophilic than the parental www.sciencedirect.com
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Figure 2
Schematic illustration of the cellular sterol trafficking pathways and sterol-binding proteins is discussed in this review. Sterol transport is indicated by thick arrows and sterol-binding by specific proteins by thin arrows. Cholesterol precursors are shown in dark green (PreC, several cholesterol precursors; Desmo, desmosterol), oxysterols in light green (OH-C, several oxysterols; 25-HC, 25-hydroxycholesterol; 7-KC, 7-ketocholesterol), cholesterol (Chol) in light blue and cholesteryl acetate (CAc) in dark blue. MACPF, membrane attack complex/perforin; ABCG1 and ABCG4, ATPbinding cassette transporters G1 and G4; NPC1 and NPC2, Niemann-Pick type C proteins 1 and 2; INSIG, insulin-induced gene; SCAP, sterol response element-binding protein cleavage activating protein; PM, plasma membrane; LE, late endosome; ER, endoplasmic reticulum. Numbers in brackets indicate the references, in which the findings on sterol trafficking/binding are reported.
compounds because of additional hydroxy- or ketogroups. Analogously to the sterol precursors, oxysterols associate with membranes and may display differential distributions between cell types. Members of the oxysterol-binding protein (OSBP) family (ORPs, OSBPrelated proteins) show distinct, though partially overlapping binding-specificities for different sterols, and evidence from yeast suggests a function for these proteins in the nonvesicular intracellular transport of sterols [25]. However, for mammalian ORPs functional studies point to more diverse roles and rather suggest that these proteins mainly act as sterol sensors [26]. At present, little is known of the trafficking of sterol precursors or oxysterols in higher eukaryotes. Lipid rafts were suggested to participate in the retrograde movement of sterol precursors from the plasma membrane to the ER as raft disruption (by cholesterol depletion) retards the conversion of precursors to cholesterol [27]. Another interpretation might be that cholesterol removal does not perturb the transport process per se but increases the affinity of sterol precursors to the plasma membrane.
active site in the ER lumen. Sterol acetylation acts as a detoxification mechanism, resulting in the secretion of the acetylated sterol via the secretory pathway. Substrate specificity of the deacetylase Say1p appears to control, which sterols are deacetylated and retained in the cell. The steroid hormone precursor pregnenolone is also acetylated but its export is independent of secretion, indicating that yeast has several mechanisms to export acetylated sterols. Importantly, sterol deacetylation is conserved in metazoans, with the human arylacetamide deacetylase capable of providing deacetylation activity when expressed in yeast [28]. In vertebrates, glucuronidation and sulfonation are known modifications for detoxification and excretion of sterols and steroids [29,30] but whether acetylation could also operate as such, remains open. Mechanistically, an interesting question is also how cholesterol acetate — that is even more hydrophobic than cholesterol — is rendered soluble in the ER lumen or in the extracellular milieu.
Novel modifications
It is evident that structural information on the interactions between sterols and proteins is crucial for understanding protein mediated sterol transport and the mechanisms by which sterols affect proteins. Also in this area, several new findings have been made.
A novel, reversible sterol modification was recently identified in S. cerevisiae, the sterol acetylation/deacetylation cycle [28]. Both the acetyltransferase and deacetylase enzymes are ER-membrane proteins, with a putative www.sciencedirect.com
Structural considerations on protein–sterol interactions
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Membrane attack complex proteins
Papers published in the same issue of Science determined the crystal structures of two membrane attack complex/ perforin (MACPF) domains and revealed a novel role for cholesterol in vertebrate immunity [31,32]. The structure of MACPF appears similar to that of the pore-forming cholesterol-dependent cytolysins (CDCs), a large family of bacterial toxins capable of generating oligomeric pores in membranes by specifically binding to cholesterol. The authors propose that MACPF proteins function in a CDC like manner. Interestingly, besides immunity MACPF proteins also play important roles in embryonic development and neuronal cell migration [33,34]. It therefore appears plausible that cholesterol is involved in regulating MACPF dependent aspects of these processes. SREBP-machinery
Recent studies by Brown and Goldstein have gained insight into the roles of cholesterol and oxysterols in regulating the transport of sterol regulatory elementbinding proteins (SREBPs) from the ER. It has been known for some time that both cholesterol and 25-hydroxycholesterol (25-HC) induce the interaction between Insig and Scap and thereby retain SREBP in the ER [35]. This prevents SREBP from being transported to and processed in the Golgi and consequently inhibits the expression of genes involved in sterol biosynthesis. It now appears that cholesterol and 25-HC regulate this pathway by binding different proteins. Cholesterol binds Scap causing Scap to bind Insig, whereas 25-HC binds Insig, causing Insig to bind Scap [36]. Structural analysis suggests that Scap-Insig binding renders the hexapeptide sorting signal (MELADL) in Scap inaccessible to COPII proteins [37], possibly by altering its location with respect to the membrane.
argue that this might be caused by the presence of other sterol binding sites (NPC1 contains a sterol-sensing domain outside loop-1) or by some nonphysiological aspects of the in vitro binding assay. In contrast to NPC1, NPC2 is a soluble late endosomal protein capable of transporting cholesterol in vitro. New structural studies demonstrate that NPC2 can bind a wide array of sterols besides cholesterol. These include cholesterol precursors, plant sterols and oxysterols [40]. This apparent promiscuity might be the consequence of a malleable binding site, as supported by the observation that apoNPC2 contains a loosely packed hydrophobic core, too small to accommodate a sterol [41]. In the presence of sterol, the pocket expands by structural alterations, including the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function [41]. Notably, the list of ligands did not include various glycolipids, phospholipids or fatty acids, suggesting a specific role for NPC2 in sterol transport [40].
Disease aspects related to sterol specificity Several recent studies highlight the importance of sterol structure in the pathogenesis of human diseases, or the respective mouse models. Mouse models have been generated for several inborn errors of cholesterol biosynthesis, such as Smith-Lemli-Opitz syndrome (caused by defective 7-dehydrocholesterol reductase, DHCR7) and lathosterolosis (caused by defective lathosterol-5-desaturase, SC5D). Recent studies in these mice revealed that the accumulating 7-dehydrocholesterol and lathosterol cause secretory granule malformation, apparently because of decreased membrane curvature [42]. The role of specific sterols has also been investigated in relation to Alzheimer’s disease, atherosclerosis and Huntington’s disease, as discussed below.
NPC proteins
DHCR24/seladin-1
NPC proteins are necessary for the exit of cholesterol from the endocytic system and their deficiency results in Niemann-Pick type C (NPC) disease. Although many groups have studied these proteins, their precise mechanisms of action in cholesterol transport remain unknown. Recent structural studies on both NPC1 and NPC2 have revealed some surprising features. NPC1 is a late endosomal membrane protein containing 13 putative transmembrane helices. Unexpectedly, this protein was encountered in a search for a membrane protein that binds 25-HC [38]. The binding site was localized to luminal loop-1, a 240 amino acid domain which shows strong sequence conservation in vertebrates and yeast [39]. Besides oxysterols (25-HC, 27-HC, 24(S)-HC) this site was shown to bind cholesterol, albeit less avidly than 25-HC [38]. Surprisingly, a point mutant that abolished binding of 25-HC and cholesterol was nevertheless able to revert the NPC phenotype (the accumulation of cholesterol in late endocytic organelles) [39]. The authors
DHCR24 is an oxidoreductase that catalyzes desmosterol-cholesterol conversion. Interestingly, the same protein has also been described in a different context: to be downregulated in the affected areas of Alzheimer’s disease brain (therefore named seladin-1 for selective Alzheimer’s disease indicator-1 [43]) and to confer resistance against oxidative stress and apoptotic cell death [43– 45]. Until recently, it has been difficult to reconcile the cholesterol biosynthetic and anti-apoptotic roles of the protein. However, new observations by Kuehnle et al. [46] clarify a number of issues. They show that DHCR24/seladin-1 is upregulated in an acute response and downregulated in a chronic response to oxidative stress and that the protective effects against apoptosis are observed in both cases. The acute protective effect seems to be related to cholesterol production and the fact that cholesterol offers better protection against oxidative apoptotic insult than desmosterol (possibly by counteracting lipid peroxidation and maintaining membrane
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integrity). By contrast, the prosurvival impact of low DHCR24/seladin-1 levels appears to be mediated by the tumor suppressor protein p53 in a manner that is independent of DHCR24 activity, as also reported earlier [47], and thus unrelated to membrane cholesterol concentration. Seladin-1 binding to p53 is known to result in p53 accumulation via its protection from proteasomal degradation [47]. Conversely, lowered seladin-1 levels provide deficient stabilization of p53 and therefore protect cells from p53 mediated apoptotic death [46]. Interestingly, the protective effect of DHCR24/seladin1 in Alzheimer’s disease has been linked to its role in as DHCR24 in cholesterol synthesis, via increased recruitment of cholesterol and detergent-resistant membrane (DRM) proteins into rafts and consequent reduction in beta-amyloid generation [48]. Whether this might also involve the seladin-1 function of the protein is an open question.
reporting similar observations in another HD mouse model and more remarkably, show that mice overexpressing wild-type huntingtin display higher activity of the cholesterol biosynthetic pathway and also higher levels of brain and plasma cholesterol, cholesterol precursors and 24(S)-HC with respect to wild-type animals. This suggests that normal huntingtin may play a role in cholesterol biosynthesis [54]. Intriguingly, in another study cholesterol was found to accumulate in brains and cultured neurons expressing mutant huntingtin [55]. This was linked to caveolin-1 interaction and inhibition of clathrin-independent (potentially caveolar) endocytosis by mutant huntingtin. How these findings can be reconciled with the results above is not clear, but perhaps the different mouse models and time-points of analysis contribute. In any case, both data sets agree with the conclusion that brain sterol homeostasis is perturbed in HD.
ABC-transporters
For the past years, there has been increasing appreciation on the critical role of ABC-transporters in cholesterol trafficking, particularly as proteins functioning in the ATP-dependent release of cholesterol from cells to high-density lipoproteins (HDL) or its major apoprotein ApoA-I [49]. Recent reports reveal not only that these proteins are involved in the efflux of sterol precursors and oxysterols, but also that individual transporters exhibit specificity for select sterols. The efflux of 7-ketocholesterol, the major oxysterol of oxidized low-density lipoproteins (oxLDL) and atherosclerotic lesions, is entirely dependent on the ABCG1 protein in macrophages [50]. Surprisingly, the ABCA1 transporter that is critical for cholesterol efflux to ApoA-I, is not involved. Accordingly, protection of macrophages from oxLDL-induced apoptosis specifically requires ABCG1 and reflects its ability to promote oxysterol efflux from macrophages. In astrocytes, on the contrary, the active transport of desmosterol to HDL is dependent on the overlapping functions of ABCG1 and ABCG4 transporters [51]. Huntingtin
In the central nervous system, cholesterol is synthesized locally and eliminated by conversion to an oxysterol, 24(S)-HC, that crosses the blood–brain barrier [52]. There seems to be an increasing number of neurodegenerative diseases, in which the brain sterol balance is implicated. An example is Huntington’s disease (HD) caused by an expanded trinucleotide repeat in the huntingtin gene. Cholesterol biosynthetic pathway was shown to be altered in fibroblasts and brains of HD patients as well as in a HD mouse model [53]. This seems to be attributable to a mutant huntingtin-dependent decrease in the translocation of the active SREBP to the nucleus [53]. Recent data strengthen the case by www.sciencedirect.com
Conclusions and perspectives It is becoming increasingly evident that besides their main structural membrane sterol, eukaryotic cells use a variety of minor sterol metabolites to generate specificity to sterol trafficking and protein–sterol interactions. Clearly, this characteristic is not limited to cell types that are traditionally considered as sterol processing, such as steroidogenic cells — that produce steroid hormones from cholesterol — or hepatocytes — that use cholesterol to generate bile acids. Several sterol-interacting proteins have broader specificities for sterol binding than initially anticipated, at least in in vitro settings. In living cells where kinetic and steric considerations generate more restrictions, the physiologically relevant interactions are probably more limited. The growing awareness of a multitude of sterol species in cells has important implications. It highlights the relevance of not only resolving them but also of establishing new tools to follow their cellular itineraries.
Acknowledgements The authors would like to thank Matts Linder and Aino Mutka for discussions. Our work was supported by the Academy of Finland, Sigrid Juselius Foundation, Finnish Heart Foundation and Helsinki Biomedical Graduate School.
References and recommended reading Papers of particular interest, published within the period of the review, have been highlighted as: of special interest of outstanding interest 1.
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