Proteomics

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Imipridones cause cellular toxicity in human cells and bacteria by ectopic activation of the ClpP protease


ABSTRACT: Ectopic activation of the conserved ClpP protease by chemical activators causes toxicity in bacteria and human cells due to unrestrained proteolysis. The imipridone ONC201 has been ascribed multiple mechanisms of action and is currently in cancer clinical trials. To systematically investigate the genetic dependencies of imipridone action, we screened a genome-wide CRISPR knockout library in the presence of ONC201 and its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP conferred strong resistance to both compounds, consistent with recent reports that ONC201 directly activates CLPP in cancer cells. Biochemical assays and surrogate genetic assays in yeast confirmed activation of CLPP in the absence of its regulatory subunits. Imipridone toxicity was bypassed by loss of only one other gene, the mitochondrial intermediate peptidase MIPEP, which we showed is necessary for proteolytic maturation of a CLPP precursor form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial proteins as well as cell cycle regulators. Prompted by the conservation of ClpP across kingdoms, we showed that the imipridones activate Escherichia coli ClpP in vitro and Staphylococcus aureus ClpP in a surrogate yeast assay. ONC212 and acyldepsipeptide (ADEP)-4, a known activator of bacterial ClpP, caused similar proteomic degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, Bacillus subtilis, Enterococcus faecium) and Gram-negative species (E. coli, Neisseria gonorrhoeae). Moreover, a combination of ONC212 and rifampicin eradicated antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Staphylococcus Aureus

TISSUE(S): Cell Culture

SUBMITTER: Eric Bonneil  

LAB HEAD: Mike Tyers

PROVIDER: PXD016119 | Pride | 2020-03-26

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
MT57_59_peptides_1_1_0.mzid.gz Mzid
MT60_62peptides_1_1_0.mzid.gz Mzid
MT79_82_peptides_1_1_0.mzid.gz Mzid
NALM6_peptides_1_1_0.mzid.gz Mzid
Service_Tyers_280519_10a.mgf Mgf
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Publications


Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease  ...[more]

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