Dataset Information

Ubiquitination in treated T. cruzi parasites

ABSTRACT: T. cruzi epimastigotes in the logarithmic growth phase (3×106 cells mL-1) were incubated for 12h with bortezomib (1.8 µM), GNF6702 (2.9 µM) or compound 1 (24 µM), equivalent to 8× the EC50 values of each compound. Controls were incubated in the presence of diluent (DMSO). Cells were harvested by centrifugation (1912g, 15 min, 4 °C) and washed with ice-cold PBS (1912g, 5 min, 4 °C), and finally, the cell pellets were resuspended in 1.5 mL of ice-cold lysis buffer (1 mM EDTA, 1 mM DTT, 100 μM TLCK, and 1× Roche EDTA-free cOmplete protease inhibitor cocktail in 50 mM potassium phosphate buffer, pH 7.4). Cell suspensions were submitted to 3 freeze–thaw cycles in a dry ice/ethanol bath to biologically inactivate the parasites and then lysed using the One ShotTM Cell disruptor (Constant Systems, UK) at 30 kpsi.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Trypanosoma Cruzi Dm28c

SUBMITTER: Victoriano Corpas-Lopez

LAB HEAD: Susan Wyllie

PROVIDER: PXD027524 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

Identification of a Proteasome-Targeting Arylsulfonamide with Potential for the Treatment of Chagas' Disease.

Lima Marta L ML Tulloch Lindsay B LB Corpas-Lopez Victoriano V Carvalho Sandra S Wall Richard J RJ Milne Rachel R Rico Eva E Patterson Stephen S Gilbert Ian H IH Moniz Sonia S MacLean Lorna L Torrie Leah S LS Morgillo Carmine C Horn David D Zuccotto Fabio F Wyllie Susan S

Antimicrobial agents and chemotherapy 20211004 1

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between β4 and β5 subunits that catalyze chymotrypsin-like activity. A mutation in the β5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced suscep ...[more]

PMID: 34606338

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Project description:Targeted protein degradation (TPD) has emerged as a powerful approach for removing (rather than inhibiting) proteins implicated in diseases. A key step in TPD is the formation of an induced proximity complex where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process using atomistic simulations: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) degradation efficiency via the full Cullin Ring Ligase (CRL) macromolecular assembly. We combine experimental biophysical data---in this case hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues)---with molecular dynamics (MD) simulations aided by enhanced sampling techniques to accurately predict ternary complex structures at atomic resolution. We demonstrate improved efficiency, accuracy, and reliability in the ternary structure predictions of the bromodomain of the cancer target SMARCA2 with the E3 ligase VHL mediated by three different degrader molecules. The simulations accurately reproduce X-ray crystal structures -- including a new structure that we determined in this work (PDB ID: 7S4E) -- with root mean square deviations (RMSD) of 1.1 to 1.6 \r{A}. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex. Snapshots from these simulations are used as seeds for additional simulations, where we perform 7.1 milliseconds of aggregate simulation time using Folding@home. The detailed free energy surface captures the crystal structure conformation within a low-energy basin and is consistent with solution-phase experimental data (HDX-MS and SAXS). Finally, we graft a structural ensemble of the ternary complexes onto the full CRL and perform enhanced sampling simulations, which suggest that differences in degradation efficiency may be related to the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. Several of the predicted ubiquitinated lysine residues have been validated through a ubiquitin mapping proteomics experiment. DOI 10.1038/s41467-022-33575-4

Dataset Information

Ubiquitination in treated T. cruzi parasites

Publications

Identification of a Proteasome-Targeting Arylsulfonamide with Potential for the Treatment of Chagas' Disease.

Similar Datasets

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