8 research outputs found
Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment
Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking
Post-Lysosomal Cholesterol Trafficking: Novel Tools and Insights
Cholesterol is an essential mammalian lipid. It is a major component of cellular membranes, a precursor molecule for the synthesis of hormones and bile acids, and a regulator of protein function. Although cholesterol is synthesized, de novo, in the endoplasmic reticulum, cells principally meet cholesterol requirements through uptake of lipoprotein particles. Lipoprotein-derived cholesterol is transported to the lysosome where it is transferred from the soluble lysosomal protein, NPC2, to limiting-lysosomal membrane protein NPC1. Cholesterol is then re-distributed to other cellular membranes in order to fulfill organellar cholesterol requirements; however, the cellular machineries involved in coordinating this distribution are poorly characterized. In the absence of NPC proteins, cholesterol is inefficiently redistributed beyond the lysosome, and instead accumulates, leading to lysosomal dysfunction and eventual cell death. NPC protein deficiency underlies Niemann-Pick Type C disease, a fatal neurodegenerative condition for which there is no FDA-approved treatment. While some potential therapeutics have been identified, their mechanisms remain unknown. NPC1-dependent and independent post-lysosomal cholesterol trafficking has been difficult to study, in part, because existing cholesterol probes either fail to mimic authentic cholesterol trafficking or cannot be efficiently used to identify candidate trafficking proteins. In this thesis, I describe new tools and methodologies for the study of post-lysosomal cholesterol trafficking and demonstrate their utility in NPC1-dependent and -independent cell models. First, through rigorous biochemical characterization, I evaluate a series of cholesterol probes that are functionalized to enable tagging and enrichment of protein binding partners for identification by mass spectrometry. By screening many probes, I identify permissible and non-permissible sites for functional modification. Next, I develop a strategy for delivering cholesterol probe to the lysosome in order to identify the post-lysosomal trafficking interactome. Finally, I use isotopically labeled cholesterol to investigate drug-induced cholesterol trafficking in NPC1-deficient cell models. The new tools developed in these studies will advance our understanding of post-lysosomal cholesterol trafficking and may provide mechanistic insight into therapeutics for NPC disease
Comparison of outcomes following thoracoscopic versus thoracotomy closure for persistent patent ductus arteriosus
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Lysosomal cholesterol activates mTORC1 via an SLC38A9–Niemann-Pick C1 signaling complex
The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that becomes activated at the lysosome in response to nutrient cues. Here, we identify cholesterol, an essential building block for cellular growth, as a nutrient input that drives mTORC1 recruitment and activation at the lysosomal surface. The lysosomal transmembrane protein, SLC38A9, is required for mTORC1 activation by cholesterol through conserved cholesterol-responsive motifs. Moreover, SLC38A9 enables mTORC1 activation by cholesterol independently from its arginine-sensing function. Conversely, the Niemann-Pick C1 (NPC1) protein, which regulates cholesterol export from the lysosome, binds to SLC38A9 and inhibits mTORC1 signaling through its sterol transport function. Thus, lysosomal cholesterol drives mTORC1 activation and growth signaling through the SLC38A9-NPC1 complex