RaftProt V2: understanding membrane microdomain function through lipid raft proteomes

Abstract

Cellular membranes feature dynamic submicrometer-scale lateral membrane domainsvariously referred to as lipid rafts, membrane rafts or glycosphingolipid-enriched microdomains (GEM). In order to understand the molecular functions of lipid rafts, numerous studies have utilized various biochemical methods to isolate and examine the protein composition of membrane rafts. However, interpretation of individual raft proteomics studies are confounded by the limitations of isolation methods and the dynamic nature of rafts. Knowledge-based approaches can facilitate biological data interpretation by integrating experimental evidence from existing studies. To this end, we previously developed RaftProt (http://lipid-raft-database.di.uq.edu.au/), a searchable database of mammalian lipid raft-associated proteins. Despite being a valuable and highly used resource, improvements in search capabilities and visualisation were still needed. Here, we present RaftProt V2 (http://raftprot.org), an improved update of RaftProt, enabling interrogation and integration of datasets at the cell/tissue type and UniRef/Gene level. Besides the addition of new datasets and re-mapping of all entries to both UniProt and UniRef IDs, we have annotated the level of experimental evidence for each protein entry. The search engine now allows for multiple protein or experiment searches where correlations, interactions or overlaps can be investigated. The web-interface has been completely redesigned and offers new interactive tools for data and subset selection, correlation analysis and network visualization. Overall, RaftProt aims to advance our understanding of lipid raft function by revealing the proteomes and pathways that are associated with membrane microdomains in diverse tissue and conditions.

BCR-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics

“…S1A, B). Although a fraction of the probe might remain also in the non-raft regions, strong preference to lipid rafts was clear and also supported by our shortlisting of the most efficiently and constitutively biotinylated proteins, out of which 90% were found in the RaftProt database (Mohamed et al, 2019) (Suppl. File 2).…”

“…File 2). Almost 90% of the proteins were also found in the available RaftProt databases (Mohamed et al, 2019) (Fig. 3A, Suppl.…”

ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells

“…As ORP2 is suggested to transport cholesterol toward the PM, we wondered whether the expression of genes regulating PM cholesterol‐rich raft domains was altered in ORP2 KD HUVECs. Since lipid raft‐associated proteins as a category are not included in the pathway databases used (Wikipathway, KEGG, Reactome, and PID), we ran a separate comparison of the ORP2 KD RNAseq data against the RaftProt database, 31 available at http://raftprot.org. The analysis revealed that 69.4% of the gene IDs found in the RaftProt database were significantly affected in ORP2 KD HUVECs.…”

“…Moreover, depletion of VE‐cadherin increases angiogenic sprouting, 63,67 in agreement with our data showing an increase of tip cells and perpendicular sprouts in ORP2 −/− retina. Puzzlingly, the retinal phenotype of the ORP2 −/− animals is very subtle, although the PM cholesterol organization is reported to have major effects on VEGF signaling and angiogenesis 3,5,6,8,31,32 . A plausible explanation is the existence of several overlapping cholesterol trafficking routes from endosomes to the PM, such as a Rab8‐myosin V‐mediated vesicular pathway 68 and a recently described ORP1S‐mediated pathway 69 .…”

“…Puzzlingly, the retinal phenotype of the ORP2 −/− animals is very subtle, although the PM cholesterol organization is reported to have major effects on VEGF signaling and angiogenesis. 3,5,6,8,31,32 A plausible explanation is the existence of several overlapping cholesterol trafficking routes from endosomes to the PM, such as a Rab8-myosin V-mediated vesicular pathway 68 and a recently described ORP1S-mediated pathway. 69 Generation and characterization of an endothelial-specific KO of Osbpl2 in the future will be required to reach a detailed understanding of Osbpl2/ORP2 function in the biology of ECs.…”