Proteome Dynamics during Antibiotic Persistence and Resuscitation.

During antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here, we used sustained addition of stable isotope-labeled lysine to selectively label the proteome during hipA-induced persistence and hipB-induced resuscitation of Escherichia coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-h measurement of label incorporation allowed detection of over 500 newly synthesized proteins in viable cells, demonstrating low but widespread protein synthesis during persistence. Many essential proteins were newly synthesized, and several ribosome-associated proteins such as RaiA and Sra showed high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized the ribosome-splitting GTPase HflX and various ABC transporters, restored translation machinery, and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. IMPORTANCE While bactericidal antibiotics typically require actively growing cells to exploit their function, persister cells are slowly replicating which makes them tolerant to the lethal action of antimicrobials. Here, we used an established in vitro model of bacterial persistence based on overexpression of the paradigm toxin-antitoxin (TA) system hipA/hipB to devise a generic method for temporal analysis of protein synthesis during toxin-induced persistence and antitoxin-mediated resuscitation. Our time-resolved, 24-h measurement of label incorporation demonstrated low but widespread protein synthesis during persistence. At the onset of resuscitation, cells restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. Our study provides the first global analysis of protein synthesis in persisting and resuscitating bacterial cells, and as such, presents an unprecedented resource to study the processes governing antibiotic persistence.

Discovery of a Selective Inhibitor for the YEATS Domains of ENL/AF9.

Eleven-nineteen leukemia (ENL) contains an epigenetic reader domain (YEATS domain) that recognizes lysine acylation on histone 3 and facilitates transcription initiation and elongation through its interactions with the super elongation complex (SEC) and the histone methyl transferase DOT1L. Although it has been known for its role as a fusion protein in mixed lineage leukemia (MLL), overexpression of native ENL, and thus dysregulation of downstream genes in acute myeloid leukemia (AML), has recently been implicated as a driver of disease that is reliant on the epigenetic reader activity of the YEATS domain. We developed a peptide displacement assay (histone 3 tail with acylated lysine) and screened a small-molecule library totaling more than 24,000 compounds for their propensity to disrupt the YEATS domain-histone peptide binding. Among these, we identified a first-in-class dual inhibitor of ENL ( Kd = 745 ± 45 nM) and its paralog AF9 ( Kd = 523 ± 53 nM) and performed "SAR by catalog" with the aim of starting the development of a chemical probe for ENL.

Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial ß-barrel proteins.

Mitochondrial ß-barrel proteins are encoded in the nucleus, translated by cytosolic ribosomes, and then imported into the organelle. Recently, a detailed understanding of the intramitochondrial import pathway of ß-barrel proteins was obtained. In contrast, it is still completely unclear how newly synthesized ß-barrel proteins reach the mitochondrial surface in an import-competent conformation. In this study, we show that cytosolic Hsp70 chaperones and their Hsp40 cochaperones Ydj1 and Sis1 interact with newly synthesized ß-barrel proteins. These interactions are highly relevant for proper biogenesis, as inhibiting the activity of the cytosolic Hsp70, preventing its docking to the mitochondrial receptor Tom70, or depleting both Ydj1 and Sis1 resulted in a significant reduction in the import of such substrates into mitochondria. Further experiments demonstrate that the interactions between ß-barrel proteins and Hsp70 chaperones and their importance are conserved also in mammalian cells. Collectively, this study outlines a novel mechanism in the early events of the biogenesis of mitochondrial outer membrane ß-barrel proteins.