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Abstract. Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDLcholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using (LDL) l (8). LDL is delivered to lysosomes where the protein/phospholipid coat is degraded, and LDL-cholesteryl esters are hydrolyzed to unesterified cholesterol. LDL-derived cholesterol is used by cells to synthesize cellular membranes, bile acids, and steroid hormones (8). LDL-derived cholesterol also elicits several regulatory responses, including suppression of cholesterol biosynthesis and LDL receptor activity, as well as activation of the cholesterol-esterifying enzyme, acyl coenzyme A/cholesterol acyl transferase (ACAT) (8). The process of receptor-mediated internalization of LDL and the effects of LDL-cholesterol on cellular cholesterol metabolism have been extensively studied (8); however, little information is known about the fate of cholesterol formed by lysosomal hydrolysis of LDL-cholesteryl esters. In particular, the pathway and mechanism of intracellular transport of LDL-cholesterol from lysosomes to cellular membranes are obscure. In addition, it is not known whether the regulatory actions of LDL-cholesterol require either the transport of cholesterol to regulatory sites within cells or metabolic transformation of the sterol.Somatic cells exhibiting specific defects in LDL metabolism may be useful in delineating the mechanisms of intracellular transport of and regulation by LDL-cholesterol. A defect in LDL-cholesterol metabolism in fibroblasts from patients with Niemann-Pick disease type C (NPC) has been described by Pentchev and co-workers (25-27) and our laboratory (21). Although LDL is bound and internalized, and the cholesteryl esters hydrolyzed normally in NPC fibroblasts, LDL-derived cholesterol does not elicit normal regulatory responses (21,(25)(26)(27). In NPC cells, LDL does not stimulate cholesterol esterification, and LDL suppression of cholesterol synthesis and LDL receptor activity is impaired (21,25,27). The defective regulation of cellular cholesterol metabolism in NPC cells appears to be specific for LDL-derived cholesterol, since 25-hydroxycholesterol, a nonlipoprotein effector, does stimulate cholesterol esterification as well as downregulate cholesterol synthesis and LDL receptor activity normally in NPC cells (21). Also, exogenously added mevalonate suppresses...

The CLN6 gene that causes variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a recessively inherited neurodegenerative disease that features blindness, seizures, and cognitive decline, maps to 15q21-23. We have used multiallele markers spanning this approximately 4-Mb candidate interval to reveal a core haplotype, shared in Costa Rican families with vLINCL but not in a Venezuelan kindred, that highlighted a region likely to contain the CLN6 defect. Systematic comparison of genes from the minimal region uncovered a novel candidate, FLJ20561, that exhibited DNA sequence changes specific to the different disease chromosomes: a G-->T transversion in exon 3, introducing a stop codon on the Costa Rican haplotype, and a codon deletion in exon 5, eliminating a conserved tyrosine residue on the Venezuelan chromosome. Furthermore, sequencing of the murine homologue in the nclf mouse, which manifests recessive NCL-like disease, disclosed a third lesion-an extra base pair in exon 4, producing a frameshift truncation on the nclf chromosome. Thus, the novel approximately 36-kD CLN6-gene product augments an intriguing set of unrelated membrane-spanning proteins, whose deficiency causes NCL in mouse and man.

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.

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