Dataset Information

GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver.

ABSTRACT: Abnormal cholesterol metabolism is linked to many neurodegenerative disorders. Here, we present a protocol for the production of a recombinant protein consisting of a Glutathione-S-Transferase tag fused with the Perfringolysin O (PFO). The GST-PFO tag enables analysis of the localization of cholesterol in subcellular membranes of human and mice brain and liver tissues. We have used this approach for samples from Niemann-Pick type C disease and non-alcoholic steatohepatitis models. The construct may also have applications for the diagnosis of cholesterol-accumulating disorders. For complete details on the use and execution of this protocol, please refer to Kwiatkowska et al. (2014).

SUBMITTER: Goicoechea L

PROVIDER: S-EPMC8728529 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver.

Goicoechea Leire L Arenas Fabian F Castro Fernanda F Nuñez Susana S Torres Sandra S Garcia-Ruiz Carmen C Fernandez-Checa José C JC

STAR protocols 20211229 1

Abnormal cholesterol metabolism is linked to many neurodegenerative disorders. Here, we present a protocol for the production of a recombinant protein consisting of a Glutathione-S-Transferase tag fused with the Perfringolysin O (PFO). The GST-PFO tag enables analysis of the localization of cholesterol in subcellular membranes of human and mice brain and liver tissues. We have used this approach for samples from Niemann-Pick type C disease and non-alcoholic steatohepatitis models. The construct ...[more]

PMID: 35024626

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Project description:Biological membranes organize and compartmentalize cell signaling into discrete microdomains, a process that often involves stable, cholesterol-rich platforms that facilitate protein-protein interactions. Polarized cells with distinct apical and basolateral cell processes rely on such compartmentalization to maintain proper function. In the cochlea, a variety of highly polarized sensory and non-sensory cells are responsible for the early stages of sound processing in the ear, yet little is known about the mechanisms that traffic and organize signaling complexes within these cells. We sought to determine the prevalence, localization, and protein composition of cholesterol-rich lipid microdomains in the cochlea. Lipid raft components, including the scaffolding protein caveolin and the ganglioside GM1, were found in sensory, neural, and glial cells. Mass spectrometry of detergent-resistant membrane (DRM) fractions revealed over 600 putative raft proteins associated with subcellular localization, trafficking, and metabolism. Among the DRM constituents were several proteins involved in human forms of deafness including those involved in ion homeostasis, such as the potassium channel KCNQ1, the co-transporter SLC12A2, and gap junction proteins GJA1 and GJB6. The presence of caveolin in the cochlea and the abundance of proteins in cholesterol-rich DRM suggest that lipid microdomains play a significant role in cochlear physiology.Biological significanceAlthough mechanisms underlying cholesterol synthesis, homeostasis, and compartmentalization in the ear are poorly understood, there are several lines of evidence indicating that cholesterol is a key modulator of cochlear function. Depletion of cholesterol in mature sensory cells alters calcium signaling, changes excitability during development, and affects the biomechanical processes in outer hair cells that are responsible for hearing acuity. More recently, we have established that the cholesterol-modulator beta-cyclodextrin is capable of inducing significant and permanent hearing loss when delivered subcutaneously at high doses. We hypothesize that proteins involved in cochlear homeostasis and otopathology are partitioned into cholesterol-rich domains. The results of a large-scale proteomic analysis point to metabolic processes, scaffolding/trafficking, and ion homeostasis as particularly associated with cholesterol microdomains. These data offer insight into the proteins and protein families that may underlie cholesterol-mediated effects in sensory cell excitability and cyclodextrin ototoxicity.

Dataset Information

GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver.

Publications

GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver.

Similar Datasets

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