Dataset Information

Identification of proteins crosslinked to lysosomal sphingosine and cholesterol probes

ABSTRACT: HeLa cells were labelled with lysosomally localized photocrosslinkable and clickable probes for sphingosine and cholesterol. Upon photocrosslinking and cell lysis, the protein-lipid complexes were clicked to biotin azide and enriched via Streptavidin-immunoprecipitation. Enriched protein-lipid complexes were then analyzed by LC-MS/MS to identify proteins interacting with sphingosine and cholesterol, respectively.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Frank Stein

LAB HEAD: Dr. Doris Hoeglinger

PROVIDER: PXD039780 | Pride | 2023-02-07

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	F100207.dat	Other
	F100220.dat	Other
	Lara_200814_P1371_PH_DH_Chol_5prc_TMT9plex_R1.raw	Raw
	Lara_200814_P1371_PH_DH_Sph_5prc_TMT9plex_R1.raw	Raw
	P1371_Lara_200814_P1371_PH_DH_Chol_5prc_TMT9plex_R1_results_20200901_1106_peptides.txt	Txt

Items per page:

1 - 5 of 11

Publications

Lysosome-targeted multifunctional lipid probes reveal the sterol transporter NPC1 as a sphingosine interactor.

Altuzar Janathan J Notbohm Judith J Stein Frank F Haberkant Per P Hempelmann Pia P Heybrock Saskia S Worsch Jutta J Saftig Paul P Höglinger Doris D

Proceedings of the National Academy of Sciences of the United States of America 20230309 11

Lysosomes are catabolic organelles involved in macromolecular digestion, and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases, many of which have lipid accumulation phenotypes. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and c ...[more]

PMID: 36893262

Similar Datasets

Project description:Ubash3b, also known as suppressor of T-cell receptor signaling or Sts-1, is an ill-studied atypical tyrosine phosphatase with ubiquitin binding ability. In our previous study, we hypothesized that Ubash3b plays an inhibitory role in BCR-ABL signaling through binding and dephosphorylating BCR-ABL and its interactors. The Hantschel lab recently solved the crystal structures of the p210 PH and DH domains, which are absent in the p190 variant, and demonstrated that loss-of-function mutations in the PH domain altered BCR-ABL localization, thereby reducing the interaction between Ubash3b and p2104. Taken together, this suggests differential subcellular localization of Ubash3b as a mechanism by which it interacts more strongly with p201 as compared to p190. To better understand the global impact Ubash3b has on p210, its direct kinase substrates and proteins in its phosphotyrosine signaling network, we have taken an integrative approach by combining global phosphotyrosine profiling, proximity-dependent biotinylation (BioID) and total protein analysis to investigate p210 signaling upon Ubash3b knockdown (KD). The BioID system was used to characterize Ubash3b function in p210 signaling by examining its interactome. Importantly, in all of our BioID experiments, we employed a newly technique that we have recently developed, Biotinylation Site Identification Technology (BioSITe), which directly identifies biotinylated peptides thereby increasing the reliability of the identified interactors. Here, we additionally used short hairpin RNA (shRNA) interference and generated Ubash3b knock-down (KD) and non-targeting control shRNA lines in Ba/F3 BirA*-p210 cells. Ubash3b expression was reduced >90 % in the KD cells and had a substantial effect on global tyrosine phosphorylation and on the interactome of p210. Of the 1,421 unique tyrosine phosphorylation sites identified from 830 proteins, 379 sites (from 286 proteins) exhibited a substantial increase (ÃƒÂ¢Ã¢â‚¬Â°Ã‚Â¥2-fold) in tyrosine phosphorylation upon Ubash3b KD cells compared to control cells. To date, the interactome of Ubash3b has not been extensively investigated, however, some examination of Ubash3b in the context p210 signaling has been undertaken. We designed constructs of C-terminal BirA* tagged full length Ubash3b and a deletion mutant lacking the UBA and SH3 domains leaving only the phosphatase domain tethered to BirA*. A comparative analysis of the core interactors of p210 from previous studies and Ubash3b interactome from the current study revealed 36 proteins that interact with both p210 and Ubash3b.

Project description:Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10 % additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.