PXD045081

PXD045081 is an original dataset announced via ProteomeXchange.

Title	Proteomics after labeling the vicinity of UNC-45 using miniTurbo and TurboID biotin ligases followed by streptavidin pull-down under non-stress and optogenetically induced mechanical muscle stress (OptIMMuS).
Description	This project includes two proximity labeling experiments, one to establish the vicinity of UNC-45 under non-stress conditions and one to examine changes in the vicinity of UNC-45 under optogenetically induced mechanical muscle stress. The first non-stress experiment consists of 15 samples after biotin-streptavidin pull-down in 5 replicates of 3 conditions (3 transgenic C. elegans strains). Transgenic C. elegans strains expressing the mutant BirA biotin ligases miniTurbo and TurboID in body wall muscle cells were used to compare a proximity-labeled strain expressing an UNC-45-miniTurbo-2xHA fusion protein in the muscle cells (PP3135 unc-119(ed4)III; hhIs241[unc-54p::unc-45::miniTurbo::2xHA; unc-119(+)]) with a strain expressing the biotin ligase TurboID-2xHA without fusion protein in muscle cells (PP3138 unc-119(ed4)III; hhIs242[unc-54p::TurboID::2xHA; unc-119(+)]) to find transient interactors of the myosin chaperone UNC-45 in muscle. As a control, we included a strain expressing a transgenic UNC-45-FLAG protein in the body wall muscle (PP1017 unc-119(ed4)III; hhIs84[unc-119(+); unc-54p::unc-45::FLAG]). Biotinylated proteins in 2.5 mg lysates of these strains were pulled down with streptavidin sepharose beads and identified by mass spectrometry. The second mechanical stress experiment consists of 10 samples after biotin-streptavidin pull-down in 5 replicates of 2 conditions. A transgenic C. elegans strain expressing the biotin ligase miniTurbo fused to UNC-45 in body wall muscle cells was crossed with a transgenic strain expressing the optogenetic channelrhodopsin mutant ChR2(C128S;H134R)-FLAG in body wall muscle cells (PP3358 hhIs241[unc-54p::unc-45::miniTurbo::2xHA; unc-119(+)]; hhIs251[myo-3p::ChR2(C128S,H134R)-FLAG::unc-54 3'UTR, Cbrunc-119(+)]). The latter transgene is used to contract the worms’ muscles by blue light illumination to induce mechanical muscle stress. The optogenetic channel is activated by adding the cofactor all-trans retinal (ATR) to its food source E. coli OP50. In this experiment, we compared proximity-labeling in the UNC-45-miniTurbo-2xHA expressing strain under conditions with ATR (L+, treatment, contraction) with proximity-labeling in the UNC-45-miniTurbo-2xHA expressing strain under conditions without ATR (L-, control, no contraction) to find transient interactors of the candidate UNC-45 in muscle under mechanical stress. Biotinylated proteins in 2.5 mg of lysates of these strains were pulled down using streptavidin sepharose beads and identified by mass spectrometry.
HostingRepository	PRIDE
AnnounceDate	2024-07-16
AnnouncementXML	Submission_2024-07-16_05:28:22.637.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Prerana Wagle
SpeciesList	scientific name: Caenorhabditis elegans; NCBI TaxID: 6239;
ModificationList	monohydroxylated residue
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2023-09-04 05:52:44	ID requested
⏵ 1	2024-07-16 05:28:23	announced

Dataset with its publication pending

submitter keyword: C. elegans, optogenetics, UNC-45, proximity labelling, mechanical stress, muscle

Thorsten Hoppe
contact affiliation	Institute for Genetics, CECAD Research Center, University of Cologne, Joseph-Stelzmann-Straße 26, 50931 Cologne, Germany
contact email	thorsten.hoppe@uni-koeln.de
lab head
Prerana Wagle
contact affiliation	CECAD Research Center
contact email	proteomics-facility@uni-koeln.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD045081
     1. Label: PRIDE project
     2. Name: Proteomics after labeling the vicinity of UNC-45 using miniTurbo and TurboID biotin ligases followed by streptavidin pull-down under non-stress and optogenetically induced mechanical muscle stress (OptIMMuS).