Monitoring and Modulating Intracellular Cholesterol Trafficking Using ALOD4, a Cholesterol-Binding Protein

Endapally, Shreya; Infante, Rodney E.; Radhakrishnan, Arun

doi:10.1007/978-1-4939-9136-5_12

Cited by 28 publications

(27 citation statements)

References 12 publications

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“…Taken together, the data suggests that spike potentially associates with a specific population of cholesterol, which is biochemically distinct from the sphingomyelin-associated lipid complexes enriched in canonical rafts ( Das et al, 2014 ; Endapally et al, 2019a ; Kinnebrew et al, 2019 ). Future studies will be needed to assess the nature of such cholesterol pools, potentially using toxin-based probes that discriminate between free and inaccessible forms ( Das et al, 2014 ; Endapally et al, 2019b ), and how each is affected by the identified lipophilic compounds ( Figure 4 ).…”

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation

Sanders

Jumper

Ackerman

et al. 2021

eLife

170

172

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Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

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Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation

Sanders

Jumper

Ackerman

et al. 2021

eLife

170

172

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“…Briefly, both proteins were expressed in Escherichia coli Rosetta(DE3)pLysS cells, purified by metal-affinity and gel-filtration chromatography and their lone cysteines labelled with Atto-647 maleimide following the manufacturer’s instructions (Sigma Aldrich, product #05316). Expression and purification of His 6 - and FLAG-tagged domain 4 of anthrolysin O (ALOD4) was carried out as described previously (Endapally et al, 2019b).…”

Section: Methodsmentioning

confidence: 99%

Cholesterol accessibility at the ciliary membrane controls hedgehog signaling

Kinnebrew

Iverson

Patel

et al. 2019

eLife

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104

144

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Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

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“…ALOD4 protein purification, labeling, and incubation ALOD4 protein was expressed, purified, and labeled based on a previously described protocol (Endapally et al, 2019). Briefly, the plasmid pALOD4 (Addgene 111026) was transformed into BL21 (DE3)pLysS competent cells (Invitrogen) (Gay et al, 2015).…”

Section: Pseudovirus Binding Assaymentioning

confidence: 99%

Cholesterol 25‐Hydroxylase inhibits SARS ‐CoV‐2 and other coronaviruses by depleting membrane cholesterol

et al. 2020

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Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has spread across the globe. SARS-CoV-2 is a highly infectious virus with no vaccine or antiviral therapy available to control the pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS-CoV-2. SARS-CoV-2 is a new member of the betacoronavirus genus like other closely related viruses including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Both SARS-CoV and MERS-CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon-stimulated genes (ISGs), cholesterol 25-hydroxylase (CH25H), is induced by SARS-CoV-2 infection in vitro and in COVID-19-infected patients. CH25H converts cholesterol to 25-hydrocholesterol (25HC) and 25HC shows broad anti-coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA-WA1/2020 SARS-CoV-2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus-cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID-19 and emerging viral diseases in the future.

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Monitoring and Modulating Intracellular Cholesterol Trafficking Using ALOD4, a Cholesterol-Binding Protein

Cited by 28 publications

References 12 publications

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation

Cholesterol accessibility at the ciliary membrane controls hedgehog signaling

Cholesterol 25‐Hydroxylase inhibits SARS ‐CoV‐2 and other coronaviruses by depleting membrane cholesterol

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