Abstract
We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). We also examined the biochemical properties of the sterol and its ester by employing several different mutant Chinese hamster ovary (CHO) cell lines with defined abnormalities in cholesterol metabolism as tools. AC mimics cholesterol in supporting the growth of a mutant cell line (M19) that requires cholesterol for growth. In normal cells, tritiated ACL present in low-density lipoprotein (LDL) was hydrolyzed and reesterified in a manner similar to tritiated cholesteryl linoleate (CL) in LDL. Also, in the mutant cell line (AC29) lacking the enzyme acyl-coenzyme A:cholesterol acyltransferase or in the mutant cell line (CT60) defective in the Niemann-Pick type C1 protein, the hydrolysis of ACL in LDL was normal, but the reesterification of the liberated AC was defective. Therefore, the metabolism of ACL in LDL is very similar to that of CL in LDL. Tritium-labeled AC delivered to intact CHO cells as a cyclodextrin complex was shown to photoaffinity label several discrete polypeptides, including caveolin-1. These results demonstrate AC as an effective reagent for studying cholesterol-protein interactions involved in intracellular cholesterol trafficking.
Highlights
We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL), as well as a method to label AC with 3H at high-specific radioactivity
The results show that the fate of low-density lipoprotein (LDL)-bound Azocholestanol linoleate (ACL) in Chinese hamster ovary (CHO) cells is very similar to that of LDL-bound cholesteryl linoleate (CL)
The chemical synthesis of AC and ACL were described in Materials and Methods
Summary
We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). To study lipid-protein interactions, an effective approach has Abbreviations: AC, 7,7-azocholestanol; ACL, 7,7-azocholestanol linoleate; ACO, 7,7-azo-5␣-cholestan-3-ol oleate; BHT, 2,6-di-tert-butylp-cresol; CHO, Chinese hamster ovary; CL, cholesteryl linoleate; ER, endoplasmic reticulum; MCD, methyl -cyclodextrin; NPC1, Niemann-Pick Type C1; PCC, pyridinium chlorochromate. We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL), as well as a method to label AC with 3H at high-specific radioactivity.
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