Mitochondrial endocrinology Mitochondria as key to hormones and metabolism
Download 2.44 Mb. Pdf ko'rish
|
|
cytochrome P450 fold with an open conformation which would suggest that this enzyme might bind multiple different substrates, nevertheless, like all other mitochondrial P450’s, P450c24 exhibits a narrow substrate specificity ( Annalora et al., 2010 ). Rare muta- tions in P450c24 result in elevated circulating concentrations of 1,25(OH) 2 D, causing severe neonatal hypercalcemia ( Schlingmann et al., 2011; Dauber et al., 2012 ). References Agarwal, A.K., Auchus, R.J., 2005. Cellular redox state regulates hydroxysteroid dehydrogenase activity and intracellular hormone potency. Endocrinology 146, 2531–2538 . Alpy, F., Tomasetto, C., 2006. MLN64 and MENTHO, two mediators of endosomal cholesterol transport. Biochem. Soc. Trans. 34, 343–345 . Alpy, F., Stoeckel, M.E., Dierich, A., Escola, J.M., Wendling, C., Chenard, M.P., Vanier, M.T., Gruenberg, J., Tomasetto, C., Rio, M.C., 2001. The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein. J. Biol. Chem. 276, 4261–4269 . Alpy, F., Wendling, C., Rio, M.C., Tomasetto, C., 2002. MENTHO, a MLN64 homologue devoid of the START domain. J. Biol. Chem. 277, 50780–50787 . Annalora, A.J., Goodin, D.B., Hong, W.X., Zhang, Q., Johnson, E.F., Stout, C.D., 2010. Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism. J. Mol. Biol. 396, 441–451 . Arakane, F., Sugawara, T., Nishino, H., Liu, Z., Holt, J.A., Pain, D., Stocco, D.M., Miller, W.L., Strauss 3rd., J.F., 1996. Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: implications for the mechanism of StAR action. Proc. Natl. Acad. Sci. USA 93, 13731–13736 . Arakane, F., King, S.R., Du, Y., Kallen, C.B., Walsh, L.P., Watari, H., Stocco, D.M., Strauss 3rd, J.F., 1997. Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity. J. Biol. Chem. 272, 32656– 32662 .
processing of newly synthesized steroidogenic acute regulatory protein (StAR), but not total StAR, mediates cholesterol transfer to cytochrome P450 side chain cleavage enzyme in adrenal cells. J. Biol. Chem. 276, 46583– 46596
. Auchus, R.J., Lee, T.C., Miller, W.L., 1998. Cytochrome b5 augments the 17,20 lyase activity of human P450c17 without direct electron transfer. J. Biol. Chem. 273, 3158–3165 . Baker, B.Y., Yaworsky, D.C., Miller, W.L., 2005. A pH-dependent molten globule transition is required for activity of the steroidogenic acute regulatory protein, StAR. J. Biol. Chem. 280, 41753–41760 . Baker, B.Y., Lin, L., Kim, C.J., Raza, J., Smith, C.P., Miller, W.L., Achermann, J.C., 2006. Nonclassic congenital lipoid adrenal hyperplasia: a new disorder of the steroidogenic acute regulatory protein with very late presentation and normal male genitalia. J. Clin. Endocrinol. Metab. 91, 4781–4785 . Baker, B.Y., Epand, R.F., Epand, R.M., Miller, W.L., 2007. Cholesterol binding does not predict activity of the steroidogenic acute regulatory protein, StAR. J. Biol. Chem. 282, 10223–10232 . Bayrhuber, M., Meins, T., Habeck, M., Becker, S., Giller, K., Villinger, S., Vonrhein, C., Griesinger, C., Zweckstetter, M., Zeth, K., 2008. Structure of the human voltage- dependent anion channel. Proc. Natl. Acad. Sci. USA 105, 15370–15375 . Black, S.M., Harikrishna, J.A., Szklarz, G.D., Miller, W.L., 1994. The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc. Proc. Natl. Acad. Sci. USA 91, 7247–7251 . Bose, H.S., Sugawara, T., Strauss 3rd, J.F., Miller, W.L., 1996. The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. N. Engl. J. Med. 335, 1870–1878 . Bose, H.S., Pescovitz, O.H., Miller, W.L., 1997. Spontaneous feminization in a 46XX female patient with congenital lipoid adrenal hyperplasia caused by a homozygous frame-shift mutation in the steroidogenic acute regulatory protein. J. Clin. Endocrinol. Metab. 82, 1511–1515 . Bose, H.S., Whittal, R.M., Baldwin, M.A., Miller, W.L., 1999. The active form of the steroidogenic acute regulatory protein, StAR, appears to be a molten globule. Proc. Natl. Acad. Sci. USA 96, 7250–7255 . Bose, H.S., Whittal, R.M., Huang, M.C., Baldwin, M.A., Miller, W.L., 2000. N-218 MLN64, a protein with StAR-like steroidogenic activity, is folded and cleaved similarly to StAR. Biochemistry 39, 11722–11731 . Bose, H.S., Lingappa, V.R., Miller, W.L., 2002. Rapid regulation of steroidogenesis by mitochondrial protein import. Nature 417, 87–91 . Bose, H.S., Whittal, R.M., Ran, Y., Bose, M., Baker, B.Y., Miller, W.L., 2008a. StAR-like activity and molten globule behavior of StARD6, a male germ-line protein. Biochemistry 47, 2277–2288 . Bose, M., Whittal, R.M., Miller, W.L., Bose, H.S., 2008b. Steroidogenic activity of StAR requires contact with mitochondrial VDAC1 and phosphate carrier protein. J. Biol. Chem. 283, 8837–8845 . Brandt, M.E., Vickery, L.E., 1992. Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli. Arch. Biochem. Biophys. 294, 735–740 . Brdiczka, D.G., Zorov, D.B., Sheu, S.S., 2006. Mitochondrial contact sites: their role in energy metabolism and apoptosis. Biochim. Biophys. Acta 1762, 148–163 . Brentano, S.T., Black, S.M., Lin, D., Miller, W.L., 1992. CAMP post-transcriptionally diminishes the abundance of adrenodoxin reductase mRNA. Proc. Natl. Acad. Sci. USA 89, 4099–4103 . Cali, J.J., Hsieh, C.L., Francke, U., Russell, D.W., 1991. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. J. Biol. Chem. 266, 7779–7783 . Cao, P., Bulow, H., Dumas, B., Bernhardt, R., 2000. Construction and characterization of a catalytic fusion protein system: P-45011-adrenodoxin reductase- adrenodoxin. Biochim. Biophys. Acta 1476, 253–264 . Charman, M., Kennedy, B.E., Osborne, N., Karten, B., 2010. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein. J. Lipid Res. 51, 1023–1034 . Chen, K.S., Prahl, J.M., DeLuca, H.F., 1993. Isolation and expression of human 1,25- dihydroxyvitamin D3 24-hydroxylase cDNA. Proc. Natl. Acad. Sci. USA 90, 4543–4547 . Fig. 4. Biosynthesis of vitamin D 3 . Ultraviolet light at 290–320 nm acts on the skin to cleave the B ring of 7-dehydrocholesterol to yield cholecalciferol (vitamin D 3 ). Vitamin D, is converted to 25OHD by several enzymes in the liver, principally CYP2R1. 25OHD is activated by 1 a -hydroxylation in the kidney, yielding the active hormone 1,25(OH) 2 D, which acts by binding to the vitamin D receptor (VDR). Both 25OHD and 1,25(OH) 2 D may be inactivated by P450c24 to yield 24,25(OH) 2 D or 1,24,25(OH) 3 D, respectively. 70 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 Cheng, J.B., Motola, D.L., Mangelsdorf, D.J., Russell, D.W., 2003. De-orphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxilase. J. Biol. Chem. 278, 38084–38093 . Cheng, J.B., Levine, M.A., Bell, N.H., Mangelsdorf, D.J., Russell, D.W., 2004. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc. Natl. Acad. Sci. USA 101, 7711–7715 . Cherradi, N., Chambaz, E.M., Defaye, G., 1995. Organization of 3b-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria. J. Steroid Biochem. Mol. Biol. 55, 507–514 . Cherradi, N., Rossier, M.F., Vallotton, M.B., Timberg, R., Friedberg, I., Orly, J., Wang, X.J., Stocco, D.M., Capponi, A.M., 1997. Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein and cytochrome P450scc and 3-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells. J. Biol. Chem. 272, 7899–7907 . Clark, B.J., 2012. The mammalian START domain protein family in lipid transport in health and disease. J. Endocrinol. 212, 257–275 . Clark, B.J., Wells, J., King, S.R., Stocco, D.M., 1994. The purification, cloning and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 cells mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J. Biol. Chem. 269, 28314–28322 . Dauber, A., Nguyen, T.T., Sochett, E., Cole, D.E., Horst, R., Abrams, S.A., Carpenter, T.O., Hirschhorn, J.N., 2012. Genetic defect in CYP24A1, the vitamin D 24- hydroxylase gene, in a patient with severe infantile hypercalcemia. J. Clin. Endocrinol. Metab. 97, E268–E274 . Dilworth, F.J., Black, S.M., Guo, Y.D., Miller, W.L., Jones, G., 1996. Construction of a P450c27 fusion enzyme – a useful tool for analysis of vitamin D3–25- hydroxylase activity. Biochem. J. 320, 267–271 . Dobs, A.S., Schrott, H., Davidson, M.H., Bays, H., Stein, E.A., Kush, D., Wu, M., Mitchel, Y., Illingworth, R.D., 2000. Effects of high-dose simvastatin on adrenal and gonadal steroidogenesis in men with hypercholesterolemia. Metabolism 49, 1234–1238 . Dong, Q., Miller, W.L., 2004. Vitamin D 25-hydroxylase deficiency. Mol. Genet. Metab. 83, 197–198 . Dyson, M.T., Kowalewski, M.P., Manna, P.R., Stocco, D.M., 2009. The differential regulation of steroidogenic acute regulatory protein-mediated steroidogenesis by type I and type II PKA in MA-10 cells. Mol. Cell. Endocrinol. 300, 94–103 . Edouard, T., Alos, N., Chabot, G., Roughley, P., Glorieux, F.H., Rauch, F., 2011. Short- and long-term outcome of patients with pseudo-vitamin D deficiency rickets treated with calcitriol. J. Clin. Endocrinol. Metab. 96, 82–89 . Epstein, L.F., Orme-Johnson, N.R., 1991. Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells. J. Biol. Chem. 266, 19739–19745 . Fan, J., Liu, J., Culty, M., Papadopoulos, V., 2010. Acyl-coenzyme A binding domain containing 3 (ACBD3; PAP7; GCP60): an emerging signaling molecule. Prog. Lipid Res. 49, 218–234 . Fardella, C.E., Miller, W.L., 1996. Molecular biology of mineralocorticoid metabolism. Annu. Rev. Nutr. 16, 443–470 . Feldman, D., Malloy, P.J., Miller, W.L., 2013. Genetic disorders of vitamin D synthesis and action. In: Thakker, R.V., Whyte, M.P., Eisman, J.A., Igarashi, T. (Eds.), Genetics of Bone Biology and Skeletal. Academic Press (Elsevier), London, UK, pp. 537–552 . Freeman, M.R., Dobritsa, A., Gaines, P., Segraves, W.A., Carlson, J.R., 1999. The dare gene: steroid hormone production, olfactory behavior, and neural degeneration in Drosophila. Development 126, 4591–4602 . Fu, G.K., Lin, D., Zhang, M.Y.H., Bikle, D.D., Shackleton, C.H.L., Miller, W.L., Portale, A.A., 1997a. Cloning of human 25-hydroxyvitamin D 1-hydroxylase and mutations causing vitamin D-dependent rickets type I. Mol. Endocrinol. 11, 1961–1970 . Fu, G.K., Portale, A.A., Miller, W.L., 1997b. Complete structure of the human gene for the vitamin D 1hydroxylase, P450clDNA. Cell Biol. 16, 1499–1507 . Fujieda, K., Tajima, T., Nakae, J., Sageshima, S., Tachibana, K., Suwa, S., Sugawara, T., Strauss, J.F.I.I.I., 1997. Spontaneous puberty in 46, XX subjects with congenital lipoid adrenal hyperplasia. Ovarian steroidogenesis is spared to some extent despite inactivating mutations in the steroidogenic acute regulatory protein (StAR) gene. J. Clin. Invest. 99, 1265–1271 . Gizard, F., Lavallee, B., DeWitte, F., Teissier, E., Staels, B., Hum, D.W., 2002. The transcriptional regulating protein of 132 kDa (TReP-132) enhances P450scc gene transcription through interaction with steroidogenic factor-1 in human adrenal cells. J. Biol. Chem. 277, 39144–39155 . Gucev, Z., Tee, M.K., Chitayat, D., Wherrett, D., Miller, W.L., 2013. Distinguishing deficiencies of StAR and P450scc causing neonatal adrenal failure. J. Pediatr. 162, 819–822 . Harikrishna, J.A., Black, S.M., Szklarz, G.D., Miller, W.L., 1993. Construction and function of fusion enzymes of the human cytochrome P450scc system. DNA Cell Biol. 12, 371–379 . Hasegawa, T., Zhao, L.P., Caron, K.M., Majdic, G., Suzuki, T., Shizawa, S., Sasano, H., Parker, K.L., 2000. Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol. Endocrinol. 14, 1462– 1471 .
Culty, M., Papadopoulos, V., 2005. Peripheral-type benzodiazepine receptor- mediated action of steroidogenic acute regulatory protein on cholesterol entry into Leydig cell mitochondria. Mol. Endocrinol. 19, 540–554 . Henderson, Y.C., Frederick, M.J., Jayakumar, A., Choi, Y., Wang, M.T., Kang, Y., Evans, R., Spring, P.M., Uesugi, M., Clayman, G.L., 2007. Human LBP-32/MGR is a repressor of the P450scc in human choriocarcinoma cell line JEG-3. Placenta 28, 152–160 .
Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321, 1206–1210 . Hu, M.C., Hsu, N.C., El Hadj, N.B., Pai, C.I., Chu, H.P., Wang, C.K.L., Chung, B.C., 2002. Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1. Mol. Endocrinol. 16, 1943–1950 . Huang, N., Miller, W.L., 2000. Cloning of factors related to HIV-inducible LBP proteins that regulate steroidogenic factor-1-independent human placental transcription of the cholesterol side-chain cleavage enzyme, P450scc. J. Biol. Chem. 275, 2852–2858 . Illingworth, D., Kenney, T.A., Orwoll, E.S., 1982. Adrenal function in heterozygous and homozygous hypobetalipoproteinemia. J. Clin. Endocrinol. Metab. 54, 27– 33 . Jamin, N., Neumann, J.M., Ostuni, M.A., Vu, T.K., Yao, Z.X., Murail, S., Robert, J.C., Giatzakis, C., Papadopoulos, V., Lacapere, J.J., 2005. Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor. Mol. Endocrinol. 19, 588–594 . Jo, Y., King, S.R., Khan, S.A., Stocco, D.M., 2005. Involvement of protein kinase C and cyclic adenosine 3 0 ,5 0 -monophosphate-dependent kinase in steroidogenic acute regulatory protein expression and steroid biosynthesis in Leydig cells. Biol. Reprod. 73, 244–255 . Joseph-Liauzun, E., Delmas, P., Shire, D., Ferrara, P., 1998. Topological analysis of the peripheral benzodiazepine receptor in yeast mitochondrial membranes supports a five-transmembrane structure. J. Biol. Chem. 273, 2146–2152 . Kim, C.J., Kaplan, L.E., Perwad, F., Huang, N., Sharma, A., Choi, Y., Miller, W.L., Portale, A.A., 2007. Mutations in the gene for 1-hydroxylase, CYP27B1, in patients with vitamin D 1-hydroxylase deficiency. J. Clin. Endocrinol. Metab. 92, 3177–3182 . Kim, J.M., Choi, J.H., Lee, J.H., Kim, G.H., Lee, B.H., Kim, H.S., Shin, J.H., Shin, C.H., Kim, C.J., Yu, J., Lee, D.Y., Cho, W.K., Suh, B.K., Lee, J.E., Chung, H.R., Yoo, H.W., 2011. High allele frequency of the p.Q258X mutation and identification of a novel mis-splicing mutation in the STAR gene in Korean patients with congenital lipoid adrenal hyperplasia. Eur. J. Endocrinol. 165, 771–778 . 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., 2004. Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism. J. Biol. Chem. 279, 19276–19285 . Korkhov, V.M., Sachse, C., Short, J.M., Tate, C.G., 2010. Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals. Structure 18, 677–687 . Kwon, H.J., Abi-Mosleh, L., Wang, M.L., Deisenhofer, J., Goldstein, J.L., Brown, M.S., Infante, R.E., 2009. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 137, 1213–1224 . Lacapere, J.J., Papadopoulos, V., 2003. Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis. Steroids 68, 569–585 . Lee, T.C., Miller, W.L., Auchus, R.J., 1999. Medroxyprogesterone acetate and dexamethasone are competitive inhibitors of different human steroidogenic enzymes. J. Clin. Endocrinol. Metab. 84, 2104–2110 . Leitersdorf, E., Reshef, A., Meiner, V., Levitzki, R., Schwartz, S.P., Dann, E.J., Berkman, N., Cali, J.J., Klapholz, L., Berginer, V.M., 1993. Frameshift and splice-junction mutations in the sterol 27-hydroxylase gene cause cerebrotendinous xanthomatosis in Jews or Moroccan origin. J. Clin. Invest. 91, 2488–2496 . Li, H., Yao, Z., Degenhardt, B., Teper, G., Papadopoulos, V., 2001. Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide. Proc. Natl. Acad. Sci. USA 98, 1267–1272 . Lin, D., Gitelman, S.E., Saenger, P., Miller, W.L., 1991. Normal genes for the cholesterol side chain cleavage enzyme, P450scc, in congenital lipoid adrenal hyperplasia. J. Clin. Invest. 88, 1955–1962 . Lin, D., Chang, Y.J., Strauss III, J.F., Miller, W.L., 1993. The human peripheral benzodiazepine receptor gene: cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia. Genomics 18, 643–650 . Lin, D., Sugawara, T., Strauss 3rd, J.F., Clark, B.J., Stocco, D.M., Saenger, P., Rogol, A., Miller, W.L., 1995. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267, 1828–1831 . Liu, J., Rone, M.B., Papadopoulos, V., 2006. Protein-protein interactions mediate mitochondrial cholesterol transport and steroid biosynthesis. J. Biol. Chem. 281, 38879–38893 . Lohse, P., Maas, S., Sewell, A.C., van Diggelen, O.P., Seidel, D., 1999. Molecular defects underlying Wolman disease appear to be more heterogeneous than those resulting in cholesteryl ester storage disease. J. Lipid Res. 40, 221–228 . Manna, P.R., Dyson, M.T., Stocco, D.M., 2009. Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives. Mol. Hum. Reprod. 15, 321–333 . Mannella, C.A., Forte, M., Colombini, M., 1992. Toward the molecular structure of the mitochondrial channel, VDAC. J. Bioenergy Biomembr. 24, 7–19 . Mast, N., Annalora, A.J., Lodowski, D.T., Palczewski, K., Stout, C.D., Pikuleva, I.A., 2011. Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1. J. Biol. Chem. 286, 5607–5613 . Mathieu, A.P., Fleury, A., Ducharme, L., Lavigne, P., LeHoux, J.G., 2002. Insights into steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 71
mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially unfolded states of StAR. J. Mol. Endocrinol. 29, 327–345 . McEnery, M.W., Snowman, A.M., Trifiletti, R.R., Snyder, S.H., 1992. Isolation of the mitochondrial benzodiazepine receptor: association with the voltagedependent anion channel and the adenine nucleotide carrier. Proc. Natl. Acad. Sci. USA 89, 3170–3174 . Midzak, A., Akula, N., Lecanu, L., Papadopoulos, V., 2011. Novel androstenetriol interacts with the mitochondrial translocator protein and controls steroidogenesis. J. Biol. Chem. 286, 9875–9887 . Miller, W.L., 1997. Congenital lipoid adrenal hyperplasia: the human gene knockout of the steroidogenic acute regulatory protein. J. Mol. Endocrinol. 19, 227–240 . Miller, W.L., 2005. Minireview: regulation of steroidogenesis by electron transfer. Endocrinology 146, 2544–2550 . Miller, W.L., 2007. StAR search–what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import. Mol. Endocrinol. 21, 589–601 . Miller, W.L., Auchus, R.J., 2011. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151 . Miller, W.L., Bose, H.S., 2011. Early steps in steroidogenesis: intracellular cholesterol trafficking. J. Lipid Res. 52, 2111–2135 . Muller, A., Muller, J.J., Muller, Y.A., Uhlmann, H., Bernhardt, R., Heinemann, U., 1998. New aspects of electron transfer revealed by the crystal structure of a truncated bovine adrenodoxin, Adx(4-108). Structure 6, 269–280 . Nakae, J., Tajima, T., Sugawara, T., Arakane, F., Hanaki, K., Hotsubo, T., Igarashi, N., Igarashi, Y., Ishii, T., Koda, N., Kondo, T., Kohno, H., Nakagawa, Y., Tachibana, K., Takeshima, Y., Tsubouchi, K., Strauss III, J.F., Fujieda, K., 1997. Analysis of the Download 2.44 Mb. Do'stlaringiz bilan baham: 
|