A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol

Abstract

This article is available online at http://www.jlr.org by coordinated homeostatic mechanisms, primarily involving the regulated proteolysis of the sterol regulatory element-binding protein (SREBP)-2 transcription factor in the Golgi ( 1 ). When ER sterols are abundant, SREBP-2 is retained in the ER in a complex with the cholesterol-sensing protein, SREBP cleavage-activating protein (SCAP), and the ER retention proteins, Insig-1 or -2 ( 2, 3 ). When cholesterol content in the ER is depleted, SCAP undergoes a conformational change and is released from Insig, allowing the SREBP-SCAP complex to translocate to the Golgi apparatus ( 4, 5 ) where it undergoes proteolytic maturation with release of the SREBP transcription factor ( 6 ).Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols ( 1, 7-9 ) Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are generated enzymatically and act as important regulators of cholesterol homeostasis, despite their presence in cells being only at 0.1% the concentration of cholesterol ( 9 ). At the transcriptional level, 25-HC can bind the liver X receptors (LXRs) to active LXR-mediated transcription that results in increased cholesterol effl ux and elimination ( 10, 11 ). 25-HC also inhibits SREBP maturation and subsequent transcription of genes involved in cholesterol biosynthesis and uptake ( 1 ). In contrast to cholesterol, which directly interacts with SCAP, 25-HC enhances the interaction between SCAP and Insig proteins, resulting in retention of the SREBP-2-SCAP complex in the ER ( 12, 13 ).

Sterol Metabolism and Transport in Atherosclerosis and Cancer

“…When cells are incubated with 25-HCTL in the presence of LDL, 25-HCTL enters cells via LDLR-mediated endocytosis, and is transported to the LE/LYS. Like cholesterol, egress of 25-HCTL from the intracellular compartments requires functional NPC1 protein ( 120 ), which is consistent with 25-OHC-binding ability of NPC1 protein ( 119 ). Furthermore, 25-HCTL can be transported to the ER, and is also redistributed to the PM and the recycling endosome ( 120 ).…”

“…Like cholesterol, egress of 25-HCTL from the intracellular compartments requires functional NPC1 protein ( 120 ), which is consistent with 25-OHC-binding ability of NPC1 protein ( 119 ). Furthermore, 25-HCTL can be transported to the ER, and is also redistributed to the PM and the recycling endosome ( 120 ). It remains to be determined how endogenously synthesized oxysterols are transported from the sites where they are synthesized: the ER and mitochondria.…”

“…Recent work developed an intrinsically fluorescent oxysterol, 25-hydroxycholestatrienol (25-HCTL) that mimics 25-OHC ( 120 ). 25-HCTL is a hydroxylated derivative of cholestatrienol (CTL), a fluorescent cholesterol analog without fluorophore that may affect the physical properties of cholesterol.…”

Fluorescent Sterols and Cholesteryl Esters as Probes for Intracellular Cholesterol Transport

“…51,73,[101][102][103] Very recently, the synthesis of a side-chain hydroxylated derivative of CTL has been reported and applied for characterizing transport and metabolism of 25-hydroxycholesterol. 104 This 25-hydroxy-CTL is imported into cells by LDL-mediated endocytic uptake and modulates sterol homeostatic responses in a manner similar to 25-hydroxycholesterol. This confirms that P-sterols mimic their natural counterparts very closely, making them very good probes for the analysis of sterol metabolism and transport.…”

Pathways and Mechanisms of Cellular Cholesterol Efflux—Insight From Imaging

“…In a pioneering study, Iaea et al (2015) presented a novel intrinsically fluorescent analogue of 25-hydroxycholesterol, which contains only two additional double bonds in the ring system compared to the parent oxysterol ( Iaea et al, 2015 ). This analogue named 25-hydroxycholestatrienol can suppress cholesterol synthesis, activate ACAT for sterol esterification, be esterified in cells and stimulate expression of ABCA1 via activation of LXRs ( Iaea et al, 2015 ). Its export from lysosomes depends on NPC1 and to a lower extent on NPC2 ( Iaea et al, 2015 ; Petersen et al, 2020 ).…”