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STARTing to understand MLN64 function in cholesterol transport

2010; Elsevier BV; Volume: 51; Issue: 8 Linguagem: Inglês

10.1194/jlr.e008854

1539-7262

Attilio Rigotti, David E. Cohen, Silvana Zanlungo,

Alzheimer's disease research and treatments

MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 proteinJournal of Lipid ResearchVol. 51Issue 5PreviewNiemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder, caused in most cases by mutations in the late endosomal protein NPC1. A hallmark of NPC disease is endosomal cholesterol accumulation and an impaired cholesterol homeostatic response, which might affect cholesterol transport to mitochondria and, thus, mitochondrial and cellular function. This study aimed to characterize mitochondrial cholesterol homeostasis in NPC disease. Using wild-type and NPC1-deficient Chinese hamster ovary cells, stably transfected with a CYP11A1 complex to assess mitochondrial cholesterol import by pregnenolone production, we show that cholesterol transport to the mitochondrial inner membrane is not affected by loss of NPC1. Full-Text PDF Open Access In most cell types, intracellular transport mechanisms that mediate cholesterol movement into the mitochondrial outer and inner membranes are not well understood. By contrast in steroidogenic tissues, the steroidogenic acute regulatory (StAR) protein in conjunction with the benzodiazepine receptor have been defined as key mediators of cholesterol flux toward the mitochondrial inner membrane (1.Lavigne P. Najmanivich R. Lehoux J.G. Mammalian StAR-related lipid transfer (START) domains with specificity for cholesterol: structural conservation and mechanism of reversible binding.Subcell. Biochem. 2010; 51: 425-437Crossref PubMed Scopus (43) Google Scholar, 2.Alpy F. Tomasetto C. Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.J. Cell Sci. 2005; 118: 2791-2801Crossref PubMed Scopus (290) Google Scholar). Furthermore, StAR-mediated cholesterol transfer is critical for steroid synthesis, and deficiencies in this protein are responsible for the rare but severe disorder of human steroidogenesis known as congenital lipoid adrenal hyperplasia (1.Lavigne P. Najmanivich R. Lehoux J.G. Mammalian StAR-related lipid transfer (START) domains with specificity for cholesterol: structural conservation and mechanism of reversible binding.Subcell. Biochem. 2010; 51: 425-437Crossref PubMed Scopus (43) Google Scholar, 2.Alpy F. Tomasetto C. Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.J. Cell Sci. 2005; 118: 2791-2801Crossref PubMed Scopus (290) Google Scholar). A family of genes has now been identified based upon predicted structural homology to the StAR protein (3.Ponting C.P. Aravind L. START: a lipid-binding domain in StAR, HD-ZIP and signalling proteins.Trends Biochem. Sci. 1999; 24: 130-132Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Each protein contains a ∼245 amino acid StAR-related lipid transfer (START) domain. The mammalian genome contains 15 START domain proteins (StARD1–StARD15) with StARD1 being a synonym for StAR (4.Soccio R.E. Breslow J.L. StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism.J. Biol. Chem. 2003; 278: 22183-22186Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Considering that five START domain proteins have been demonstrated to bind cholesterol (1.Lavigne P. Najmanivich R. Lehoux J.G. Mammalian StAR-related lipid transfer (START) domains with specificity for cholesterol: structural conservation and mechanism of reversible binding.Subcell. Biochem. 2010; 51: 425-437Crossref PubMed Scopus (43) Google Scholar, 2.Alpy F. Tomasetto C. Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.J. Cell Sci. 2005; 118: 2791-2801Crossref PubMed Scopus (290) Google Scholar), this opens the possibility that some members of this protein family may be involved in cholesterol transport to the mitochondria in nonsteroidogenic cells in which they are expressed. If true, START domain proteins could mediate the synthesis of additional cholesterol-derived products such as oxysterols, neurosteroids, and bile acids. MLN64 (a.k.a. StARD3) exhibits some features suggesting its role in mitochondrial cholesterol delivery. It contains an N-terminal transmembrane domain called MENTAL (MLN64 N-terminal) in addition to its C-terminal START domain, which binds cholesterol at a 1:1 ratio (5.Tsujishita Y. Hurley J.H. Structure and lipid transport mechanism of a StAR-related domain.Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (450) Google Scholar). MLN64 was proposed to participate in steroidogenesis in tissues that do not express StAR, such as the placenta (6.Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss 3rd, J.F. MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 8462-8467Crossref PubMed Scopus (205) Google Scholar). However, the full-length protein has negligible pro-steroidogenic activity, and only a mutant form containing the isolated START domain significantly promotes steroid hormone synthesis via mitochondrial P450scc (6.Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss 3rd, J.F. MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 8462-8467Crossref PubMed Scopus (205) Google Scholar). Moreover, mice with targeted mutation of the MLN64 START domain appear normal and show no defect in steroidogenesis (7.Kishida T. Kostetskii I. Zhang Z. Martinez F. Liu P. Walkley S.U. Dwyer N.K. Blanchette-Mackie E.J. Radice G.L. Strauss 3rd, J.F. Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism.J. Biol. Chem. 2004; 279: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Interestingly, MLN64 is tethered to the membrane of late endosomes through its MENTAL domain, leaving the C-terminal START domain oriented toward the cytoplasm (8.Alpy F. Wendling C. Rio M.C. Tomasetto C. MENTHO, a MLN64 homologue devoid of the START domain.J. Biol. Chem. 2002; 277: 50780-50787Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Considering that the MENTAL domain also binds cholesterol (9.Alpy F. Latchumanan V.K. Kedinger V. Janoshazi A. Thiele C. Wendling C. Rio M.C. Tomasetto C.J. Functional characterization of the MENTAL domain.J. Biol. Chem. 2005; 280: 17945-17952Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), it has been suggested that MLN64 may capture cholesterol that is released from lipoproteins within the endolysosomal compartment and transfer this cholesterol to cytosolic acceptors via its cytoplasmic START domain (2.Alpy F. Tomasetto C. Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.J. Cell Sci. 2005; 118: 2791-2801Crossref PubMed Scopus (290) Google Scholar). The localization and topology of MLN64 in late endosomes strongly suggests that this protein participates in intracellular cholesterol traffic delivered by the endocytic pathway for further metabolism in mitochondria. Niemann Pick Type C (NPC) 1, another late endosome transmembrane protein, is also involved in intracellular cholesterol transport, and its deficiency leads to cholesterol accumulation in lysosomes and to the neurodegenerative Niemann Pick type C disease (10.Kwon H.J. Abi-Mosleh L. Wang M.L. Deisenhofer J. Goldstein J.L. Brown M.S. Infante R.E. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.Cell. 2009; 137: 1213-1224Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). However, a functional relationship between MLN64 and NPC1 protein has yet to be demonstrated. The paper by Charman et al. (11.Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), "MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein" in the May, 2010 issue of the Journal of Lipid Research nicely demonstrates that MLN64 acts independently of NPC1 in mediating cholesterol transport to the mitochondria. In this study, the authors exploited a classic assay for analyzing cholesterol import into mitochondria that is based on pregnenolone production in Chinese hamster ovary (CHO) cells transfected with a fusion protein containing P450scc. In this manner, they showed that cholesterol transport to the mitochondrial inner membrane was not affected by NPC1 deficiency either using cells harboring NPC1 mutations or cells in which NPC1 was knocked down using siRNA technology. Moreover, Charman et al. (11.Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) demonstrated that cholesterol transport to the mitochondrial inner membrane increased upon exposure of cholesterol-deprived cells to LDL, indicating preferential transport of endosomal cholesterol to mitochondria under these conditions. Knockdown of MLN64 by siRNA reduced cholesterol transport to the mitochondrial inner membrane in both wild-type and NPC1 deficient cells, demonstrating a significant role for MLN64 in the mitochondrial delivery of cholesterol. Interestingly, mitochondrial cholesterol contents were higher in CHO cells with loss of NPC1 compared with wild-type cells, a finding that is consistent with similar results previously reported in NPC1 neurons and hepatocytes (12.Yu W. Gong J.S. Ko M. Garver W.S. Yanagisawa K. Michikawa M. Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function.J. Biol. Chem. 2005; 280: 11731-11739Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13.Marí M. Caballero F. Colell A. Morales A. Caballeria J. Fernandez A. Enrich C. Fernandez-Checa J.C. García-Ruiz C. Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.Cell Metab. 2006; 4: 185-198Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 14.Fernández A. Llacuna L. Fernández-Checa J.C. Colell A. Mitochondrial cholesterol loading exacerbates amyloid beta peptide-induced inflammation and neurotoxicity.J. Neurosci. 2009; 29: 6394-6405Crossref PubMed Scopus (128) Google Scholar) and suggests preferential shunting of cholesterol to mitochondria in the absence of a functional NPC1 pathway. Consistent with this possibility, depletion of MLN64 mitigated the increase in mitochondrial cholesterol in NPC1-deficient cells. Taken together, these observations indicate that MNL64-mediated cholesterol transfer is responsible for the accumulation of mitochondrial cholesterol that is observed in NPC disease. This is pathophysiologically relevant because the increase in cholesterol content has been linked to mitochondrial dysfunction characterized by reduced glutathione content, membrane potential, and ATP synthesis (12.Yu W. Gong J.S. Ko M. Garver W.S. Yanagisawa K. Michikawa M. Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function.J. Biol. Chem. 2005; 280: 11731-11739Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13.Marí M. Caballero F. Colell A. Morales A. Caballeria J. Fernandez A. Enrich C. Fernandez-Checa J.C. García-Ruiz C. Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.Cell Metab. 2006; 4: 185-198Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 14.Fernández A. Llacuna L. Fernández-Checa J.C. Colell A. Mitochondrial cholesterol loading exacerbates amyloid beta peptide-induced inflammation and neurotoxicity.J. Neurosci. 2009; 29: 6394-6405Crossref PubMed Scopus (128) Google Scholar). The findings by Charman et al. (11.Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) complement our previous report that adenovirus-mediated overexpression of MLN64 induces an increase in hepatic free cholesterol that is associated with apoptosis and liver damage (15.Tichauer J.E. Morales M.G. Amigo L. Galdames L. Klein A. Quinones V. Ferrada C. Alvarez A.R. Rio M.C. Miquel J.F. et al.Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse.World J. Gastroenterol. 2007; 13: 3071-3079Crossref PubMed Scopus (15) Google Scholar). Based on the findings of Charman et al. (11.Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), we hypothesize that the liver damage observed in MLN64-overexpressing mice is mediated, at least in part, by an increase in mitochondrial cholesterol transport. Additional studies will be required to test this hypothesis. MLN64 is expressed in all tissues (4.Soccio R.E. Breslow J.L. StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism.J. Biol. Chem. 2003; 278: 22183-22186Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 6.Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss 3rd, J.F. MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 8462-8467Crossref PubMed Scopus (205) Google Scholar), suggesting the possibility that it broadly regulates sterol metabolism. In the liver, it may be critical for cholesterol movement into mitochondria in order to initiate the acidic pathway for bile acid synthesis (16.Russell D.W. Fifty years of advances in bile acid synthesis and metabolism.J. Lipid Res. 2009; 50: S120-S125Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Indeed, two studies (17.Pandak W.M. Ren S. Marques D. Hall E. Redford K. Mallonee D. Bohdan P. Heuman D. Gil G. Hylemon P. Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes.J. Biol. Chem. 2002; 277: 48158-48164Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18.Ren S. Hylemon P. Marques D. Hall E. Redford K. Gil G. Pandak W.M. Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis.J. Lipid Res. 2004; 45: 2123-2131Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) have demonstrated that overexpression of StAR, the closest MLN64 homolog, leads to an increase in bile acid synthesis in hepatocytes. By contrast, MLN64 overexpression in primary rat hepatocytes and in mouse liver caused a rather small increase in bile acid synthesis and no change in bile acid pool size and composition (18.Ren S. Hylemon P. Marques D. Hall E. Redford K. Gil G. Pandak W.M. Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis.J. Lipid Res. 2004; 45: 2123-2131Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar; Rigotti, Cohen, and Zanlungo, unpublished observations). Clearly, additional studies are required to understand the small effect observed in bile salt synthesis by MLN64 overexpression in hepatocytes. Nevertheless, it is noteworthy that Charman et al. (11.Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) demonstrated that MLN64 depletion in CHO cells inhibited pregnenolone formation by only 30–40%, an indication that MLN64-dependent cholesterol transport is not the only pathway for mitochondrial cholesterol import in these cells. MLN64 may also be relevant for cholesterol transport into mitochondria in other tissues and important for the synthesis of 27-hydroxycholesterol, an oxysterol that regulates cholesterol metabolism and homeostasis in the mammalian brain (19.Björkhem I. Crossing the barrier: oxysterols as cholesterol transporters and metabolic modulators in the brain.J. Intern. Med. 2006; 260: 493-508Crossref PubMed Scopus (281) Google Scholar). One potential alternative route for cholesterol transport into hepatic mitochondria is via StARD4, a START domain protein that binds cholesterol and is highly expressed in the liver (2.Alpy F. Tomasetto C. Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.J. Cell Sci. 2005; 118: 2791-2801Crossref PubMed Scopus (290) Google Scholar). Overexpression of StARD4 in primary hepatocytes increases bile acid production (20.Rodriguez-Agudo D. Ren S. Wong E. Marques D. Redford K. Gil G. Hylemon P. Pandak W.M. Intracellular cholesterol transporter StarD4 binds free cholesterol and increases cholesteryl ester formation.J. Lipid Res. 2008; 49: 1409-1419Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, mice with targeted mutation for StARD4 exhibited only modest alterations in hepatic steroid metabolism (21.Riegelhaupt J.J. Waase M.P. Garbarino J. Cruz D.E. Breslow J.L. Targeted disruption of steroidogenic acute regulatory protein D4 leads to modest weight reduction and minor alterations in lipid metabolism.J. Lipid Res. 2010; 51: 1134-1143Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The findings of Charman et al. (11.Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) highlight the relevance of cholesterol transport to the mitochondria and suggest that abnormalities in this process may lead to mitochondrial dysfunction as a key pathogenic event underlying several neurodegenerative diseases. In addition to the association between increased mitochondrial cholesterol contents, reduced glutathione and ATP production in NPC1 cells, cholesterol chelation by cyclodextrin treatment of NPC1 mitochondria restores ATP synthesis and mitochondrial function (12.Yu W. Gong J.S. Ko M. Garver W.S. Yanagisawa K. Michikawa M. Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function.J. Biol. Chem. 2005; 280: 11731-11739Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Mitochondrial dysfunction appears to be a key element not only in certain neurodegenerative diseases but also in diseases associated with damage to the liver and heart (22.Mantena S.K. King A.L. Andringa K.K. Eccleston H.B. Bailey S.M. Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases.Free Radic. Biol. Med. 2008; 44: 1259-1272Crossref PubMed Scopus (344) Google Scholar, 23.Tsutsui H. Kinugawa S. Matsushima S. Mitochondrial oxidative stress and dysfunction in myocardial remodelling.Cardiovasc. Res. 2009; 81: 449-456Crossref PubMed Scopus (297) Google Scholar, 24.Moreira P.I. Zhu X. Wang X. Lee H.G. Nunomura A. Petersen R.B. Perry G. Smith M.A. Mitochondria: a therapeutic target in neurodegeneration.Biochim. Biophys. Acta. 2010; 1802: 212-220Crossref PubMed Scopus (238) Google Scholar, 25.Querfurth H.W. La F.M. Ferla F.M. Alzheimer's disease.N. Engl. J. Med. 2010; 362: 329-344Crossref PubMed Scopus (3785) Google Scholar). For instance, hepatocytes from leptin-deficient ob/ob mice or rats with diet-induced mitochondrial cholesterol accumulation exhibited reduced mitochondrial membrane fluidity and glutathione levels that correlated with increased sensitivity to TNFα-dependent cell death (13.Marí M. Caballero F. Colell A. Morales A. Caballeria J. Fernandez A. Enrich C. Fernandez-Checa J.C. García-Ruiz C. Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.Cell Metab. 2006; 4: 185-198Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). In this connection, we have previously shown increased MLN64 mRNA and protein levels in a murine model of liver damage induced by bile acids (15.Tichauer J.E. Morales M.G. Amigo L. Galdames L. Klein A. Quinones V. Ferrada C. Alvarez A.R. Rio M.C. Miquel J.F. et al.Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse.World J. Gastroenterol. 2007; 13: 3071-3079Crossref PubMed Scopus (15) Google Scholar), suggesting that MLN64-mediated cholesterol transport to mitochondria may underlie bile acid-induced cytotoxicity. In addition, mitochondrial dysfunction is commonly associated with increased generation of reactive oxygen species, induction of apoptosis, inflammation, and fibrosis, depending upon the tissue (22.Mantena S.K. King A.L. Andringa K.K. Eccleston H.B. Bailey S.M. Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases.Free Radic. Biol. Med. 2008; 44: 1259-1272Crossref PubMed Scopus (344) Google Scholar, 23.Tsutsui H. Kinugawa S. Matsushima S. Mitochondrial oxidative stress and dysfunction in myocardial remodelling.Cardiovasc. Res. 2009; 81: 449-456Crossref PubMed Scopus (297) Google Scholar, 24.Moreira P.I. Zhu X. Wang X. Lee H.G. Nunomura A. Petersen R.B. Perry G. Smith M.A. Mitochondria: a therapeutic target in neurodegeneration.Biochim. Biophys. Acta. 2010; 1802: 212-220Crossref PubMed Scopus (238) Google Scholar, 25.Querfurth H.W. La F.M. Ferla F.M. Alzheimer's disease.N. Engl. J. Med. 2010; 362: 329-344Crossref PubMed Scopus (3785) Google Scholar). Future studies will be required to determine the pathophysiological relationships between MLN64 expression/activity to abnormal mitochondrial cholesterol transport in human diseases.