Fig. 1: Development of LYMTAC as a modular tool for lysosomal degradation of membrane proteins. | Nature Communications

Fig. 1: Development of LYMTAC as a modular tool for lysosomal degradation of membrane proteins.

From: LYMTACs:chimeric small molecules repurpose lysosomal membrane proteins for target protein relocalization and degradation

Fig. 1

A Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Quantified data are representative of two independent experiments. C Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand) and PROTAC TL-12-186. D Quantitative proteomics analysis of LYMTAC-1. HEK293 cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. Differential expression was assessed with the limma moderated t-test (empirical-Bayes framework, two-sided). Reported P values are unadjusted; no correction for multiple comparisons was applied. E Quantitative proteomics analysis of PROTAC (TL-12-186). HEK293 cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM PROTAC for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. Differential expression was assessed with the limma moderated t-test (empirical-Bayes framework, two-sided). Reported P values are unadjusted; no correction for multiple comparisons was applied. F LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293 cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Quantified data are representative of three independent experiments. G LYMTAC-1 induced dose-dependent degradation of EPHA2. Quantified data are representative of three independent experiments. H, I HEK293 cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Quantified data are representative of two independent experiments.

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