Dataset Information

Identification of the target proteins for compound 1

ABSTRACT: In this project, we used biotin-labeled compound 1 to pull down proteins in U2OS cell lysates with or without the competition of free compound 1, and then to identify the cellular binding of compound 1.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yanli Liu

PROVIDER: PXD034927 | iProX | Fri Jun 24 00:00:00 BST 2022

REPOSITORIES: iProX

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Publications

A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II.

Liu Yanli Y Iqbal Aman A Li Weiguo W Ni Zuyao Z Wang Yalong Y Ramprasad Jurupula J Abraham Karan Joshua KJ Zhang Mengmeng M Zhao Dorothy Yanling DY Qin Su S Loppnau Peter P Jiang Honglv H Guo Xinghua X Brown Peter J PJ Zhen Xuechu X Xu Guoqiang G Mekhail Karim K Ji Xingyue X Bedford Mark T MT Greenblatt Jack F JF Min Jinrong J

Nature communications 20220916 1

Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target ...[more]

PMID: 36114190

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Project 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.

Dataset Information

Identification of the target proteins for compound 1

Publications

A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II.

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