PXD044921-2

PXD044921 is an original dataset announced via ProteomeXchange.

Title	A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for DNAJC13 in opioid receptor downregulation
Description	G 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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:57:38.596.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hayden Adoff
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue; alkylated residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2023-08-28 21:13:05	ID requested
1	2024-09-03 15:07:00	announced
⏵ 2	2024-10-22 06:57:40	announced	2024-10-22: Updated project metadata.

10.1038/s41589-024-01705-2;

submitter keyword: endosomal sorting, isobaric labeling, APEX2, proximity labeling,GPCR, endolysosome, quantitative proteomics, DNAJC13, opioid receptor

Braden Lobingier
contact affiliation	Department of Chemical Physiology and Biochemistry Oregon Health & Science University 3181 SW Sam Jackson Park Road, Mail Code L-334, Portland, OR, 97239, USA
contact email	lobingib@ohsu.edu
lab head
Hayden Adoff
contact affiliation	Lobingier Lab, Department of Chemical Physiology and Biochemistry, Oregon Health and Sciences University
contact email	adoff@ohsu.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/09/PXD044921

PRIDE project URI

• PRIDE
  1. PXD044921
     1. Label: PRIDE project
     2. Name: A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for DNAJC13 in opioid receptor downregulation