eprintid: 31709
rev_number: 17
eprint_status: archive
userid: 6735
dir: disk0/00/03/17/09
datestamp: 2023-10-04 08:35:14
lastmod: 2023-10-04 15:51:09
status_changed: 2023-10-04 08:35:14
type: doctoralThesis
metadata_visibility: show
creators_name: Juarez Altuzar, Janathan Michael
title: Development, characterization and application of lysosome-targeted multi-functional sphingosine and cholesterol probes
subjects: ddc-500
subjects: ddc-540
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
cterms_swd: Sphingosine
cterms_swd: Cholesterol
cterms_swd: Lipid metabolism
cterms_swd: Niemann-Pick type C
cterms_swd: Lysosomes
cterms_swd: Lysosomal storage diseases
cterms_swd: Caged lipids
cterms_swd: Cholesterol transporter
cterms_swd: Sphingosine transporter
abstract: Lysosomes are membrane-bound organelles that act as a central hub for the recycling of biomolecules derived from cellular processes such as autophagy, endocytosis, among others. Lysosomal dysfunction is often linked to severe pathologies such as lysosomal storage disorders (LSDs), which are characterized by the aberrant accumulation of substrates, such as lipids. While cholesterol efflux from lysosomes is well-understood, the transport of other biologically active lipids such as sphingosine remain unknown. This knowledge gap is attributed to a lack of functional tools to manipulate and investigate lipids within living cells and on a single organelle level. The recent development of organelle-targeted caging groups, photoaffinity labeling and in combination with biorthogonal reactions represents a valuable and non-invasive way to identify new protein interactors of single lipid species while acquiring an exquisite spatial-temporal control.
This work presents the development, characterization and application of a method to investigate the previously enigmatic export of sphingosine from lysosomes. To this end, We have synthesized lysosome-targeted photoactivatable sphingosine (Lyso- pacSph) and lysosome-targeted photoactivatable cholesterol (Lyso-pacChol) that combine existing technologies such as photoaffinity labeling and a lysosome-targeted photoremovable caging group. In this way, the lyso-probes allow their controlled release within the lysosome using a flash of light. Their remaining modifications enable the study of their trafficking and metabolism as well as the capture of their unique lysosomal interactome. Excitingly, known cholesterol transporters, such as the abundant lysosomal protein SCARB2/LIMP-2 and Niemann-Pick type C1 (NPC1) were also identified as sphingosine interactors. Additionally, I show that both proteins play similar roles in sphingosine transport from lysosomes. Absence of either protein resulted in delayed sphingosine metabolism as observed by thin-layer chromatography as well as prolonged lysosomal localization of the sphingosine probe as shown in fluorescence microscopy experiments. The latter method also allowed me to analyze the impact of an approved drug for NPC, miglustat, on subcellular sphingosine and cholesterol trafficking. Additionally, artificial elevation of sphingosine levels in WT cells created a cholesterol export defect reminiscent of NPC disease, pointing towards a direct and causative role of sphingosine in the pathobiochemistry of this disease. Overall, the developed method presents a powerful tool to investigate the actions of biologically active lipid species with subcellular precision. This will likely inspire the generation of similar tools targeting different lipids and other organelles, thus contributing to a more detailed understanding of the intricacies of lipid-mediated signaling events.
date: 2023
id_scheme: DOI
id_number: 10.11588/heidok.00031709
ppn_swb: 1860732038
own_urn: urn:nbn:de:bsz:16-heidok-317099
date_accepted: 2022-05-25
advisor: HASH(0x55fc36c979d0)
language: eng
bibsort: JUAREZALTUDEVELOPMEN20220525
full_text_status: public
citation:   Juarez Altuzar, Janathan Michael  (2023) Development, characterization and application of lysosome-targeted multi-functional sphingosine and cholesterol probes.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/31709/1/Janathan_Thesis.pdf