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PXD044921-1

PXD044921 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleA genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for DNAJC13 in opioid receptor downregulation
DescriptionG protein-coupled receptor (GPCR) activity is critically regulated by trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-receptor interactions. Here we show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). Using our GPCR-APEX2/AUR pipeline, we demonstrate that a gene identified in our screen, DNAJC13, plays a conserved role in the trafficking of activated DOR to the lysosome. To determine how depletion of DNAJC13 changes the local proteome of DOR-positive endosomes in cultured cells, we made cell lines stably expressing DOR-APEX2 or GFP-APEX2 (cytoplasmic). We then used siRNAs to knockdown DNAJC13 from DOR-APEX2 cells and compared these results to a siRNA control. Three independent biological replicates of the proximity labeling reaction were performed follow by purification of the biotinylated proteins, trypsinization, purification of peptides, TMT labeling, and detection and analysis by MS. In addition, this TMT-dataset (18-plex) contains 6 additional samples of from an independent experiment analyzing the proximity labeling of a 2xFYVE-GFP-APEX2 construct, but these samples were not included in further data analysis. We found that the DOR proximal proteome was highly enriched in endosomal proteins compared to the cytoplasmic control, and found 13 proteins significantly changed (FDR<0.1) with DNAJC13 knockdown.
HostingRepositoryPRIDE
AnnounceDate2024-09-03
AnnouncementXMLSubmission_2024-09-03_15:06:43.133.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterHayden Adoff
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListTMT6plex-126 reporter+balance reagent acylated residue; alkylated residue
InstrumentOrbitrap Fusion
Dataset History
RevisionDatetimeStatusChangeLog Entry
02023-08-28 21:13:05ID requested
12024-09-03 15:07:00announced
22024-10-22 06:57:40announced2024-10-22: Updated project metadata.
Publication List
Novy B, Dagunts A, Weishaar T, Holland EE, Adoff H, Hutchinson E, De Maria M, Kampmann M, Tsvetanova NG, Lobingier BT, An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation. Nat Chem Biol, ():(2024) [pubmed]
10.1038/s41589-024-01705-2;
Keyword List
submitter keyword: endosomal sorting, isobaric labeling, APEX2, proximity labeling,GPCR, endolysosome, quantitative proteomics, DNAJC13, opioid receptor
Contact List
Braden Lobingier
contact affiliationDepartment of Chemical Physiology and Biochemistry Oregon Health & Science University 3181 SW Sam Jackson Park Road, Mail Code L-334, Portland, OR, 97239, USA
contact emaillobingib@ohsu.edu
lab head
Hayden Adoff
contact affiliationLobingier Lab, Department of Chemical Physiology and Biochemistry, Oregon Health and Sciences University
contact emailadoff@ohsu.edu
dataset submitter
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