Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking

Feltes, McKenna; Moores, Samantha; Gale, Sarah E.; Krishnan, Kathiresan; Mydock-McGrane, Laurel; Covey, Douglas F.; Ory, Daniel S.; Schaffer, Jean E.

doi:10.1194/jlr.d091470

Cited by 18 publications

(27 citation statements)

References 32 publications

(26 reference statements)

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“…U2OS-SRA cells efficiently take up the cholesteryl ester probe reconstituted in acLDL to lysotracker positive compartments (18). In the presence of lalistat, which prevents LAL-mediated ester hydrolysis (19,20), cargo is retained in the compartment; upon inhibitor washout, cargo is released.…”

Section: Lysosomal Cholesterol Is Differentially Distributed To the Media And The Er After CD Treatmentmentioning

confidence: 99%

Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment

Feltes

Gale

Moores

et al. 2020

Journal of Lipid Research

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Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD’s mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.

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Section: Lysosomal Cholesterol Is Differentially Distributed To the Media And The Er After CD Treatmentmentioning

confidence: 99%

Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment

Feltes

Gale

Moores

et al. 2020

Journal of Lipid Research

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“…For example, initial cholesterol photoaffinity reporters from the Cravatt group enabled proteomic analysis of many known sterol-binding proteins and revealed new candidate receptors/regulators (Figure 2e) [33]. Other photoaffinity sterol reporters have also been developed to more closely resemble the structural features and activities of cholesterol (Figure 2f) [34,35]. In addition, photoaffinity reporters of bile acids have also been developed to identify and characterize their unique receptors, transporters and regulatory enzymes [36].…”

Section: Chemical Reporters For the Direct Analysis Of Metabolite-protein Interactionsmentioning

confidence: 99%

“…(e) Sterol photoaffinity reporter [33]. f) Sterol photoaffinity reporter [34][35]. g) Bile acid photoaffinity reporter [36].…”

Section: Selective Chemical Reporters For Targeted Analysis Of Metabolite-protein Interactionsmentioning

confidence: 99%

Targeted and proteome-wide analysis of metabolite–protein interactions

Tsukidate

Hang

2020

Current Opinion in Chemical Biology

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Understanding the molecular mechanisms of endogenous and environmental metabolites is crucial for basic biology and drug discovery. With the genome, proteome and metabolome of many organisms readily available, researchers now have the opportunity to dissect how key metabolites regulate complex cellular pathways in vivo. Nonetheless, characterizing the specific and functional protein targets of key metabolites associated with specific cellular phenotypes remains a major challenge. Innovations in chemical biology are now poised to address this fundamental limitation in physiology and disease. In this review, we highlight recent advances in chemoproteomics for targeted and proteome-wide analysis of metabolite-protein interactions that have enabled the discovery of unpredicted metabolite-protein interactions and facilitated the development of new small molecule therapeutics.

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“…Direct visualization of cholesterol using fluorescent cholesterol probes and their further development together with the development of microscopic techniques enhancing their sensitivity show great promise. For that purpose various cholesterol analogues 31 – 38 , sterol binding toxins 39 , 40 , and anti-cholesterol antibodies 41 have been already analysed. However, most of the probes developed so far have not yet displayed all the appropriate characteristics for faithful monitoring of cholesterol transport 42 , 43 .…”

Section: Introductionmentioning

confidence: 99%

Sterolight as imaging tool to study sterol uptake, trafficking and efflux in living cells

Králová

Popr

Valečka

et al. 2022

Sci Rep

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Information about cholesterol subcellular localization and transport pathways inside cells is essential for understanding and treatment of cholesterol-related diseases. However, there is a lack of reliable tools to monitor it. This work follows the fate of Sterolight, a BODIPY-labelled sterol, within the cell and demonstrates it as a suitable probe for visualization of sterol/lipid trafficking. Sterolight enters cells through an energy-independent process and knockdown experiments suggest caveolin-1 as its potential cellular carrier. Intracellular transport of Sterolight is a rapid process, and transfer from ER and mitochondria to lysosomes and later to lipid droplets requires the participation of active microtubules, as it can be inhibited by the microtubule disruptor nocodazole. Excess of the probe is actively exported from cells, in addition to being stored in lipid droplets, to re-establish the sterol balance. Efflux occurs through a mechanism requiring energy and may be selectively poisoned with verapamil or blocked in cells with mutated cholesterol transporter NPC1. Sterolight is efficiently transferred within and between different cell populations, making it suitable for monitoring numerous aspects of sterol biology, including the live tracking and visualization of intracellular and intercellular transport.

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Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking

Cited by 18 publications

References 32 publications

Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment

Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment

Targeted and proteome-wide analysis of metabolite–protein interactions

Sterolight as imaging tool to study sterol uptake, trafficking and efflux in living cells

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