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System-wide analyses reveal essential roles of N-terminal protein modification in bacterial physiology

ABSTRACT: Removal of the N-terminal formyl group on nascent proteins by peptide deformylase (PDF) is the most prevalent protein modification in bacteria that impacts over 90% of the proteome. PDF is essential and a critical target of antibiotic development; however, its role in bacterial physiology remains a long-standing question. In this work, we used system-wide and time-resolved analyses of the E. coli translatome and proteome to investigate the consequences of PDF inhibition at different stages. We found that PDF inhibition results in the rapid activation of cellular stress responses, especially those associated with defects in protein folding and perturbations at the bacterial inner membrane, followed by a global downregulation of metabolic pathways. Functional assays reveal the rapid hyperpolarization of the plasma membrane and impaired membrane integrity upon PDF inhibition, suggesting that disruption of plasma membrane is the most immediate and primary consequence of formyl group retention on nascent proteins. Our work resolves the physiological function of a ubiquitous N-terminal protein modification and uncovers its crucial role in maintaining the proper structure and function of the bacterial membrane.

ORGANISM(S): Escherichia coli

PROVIDER: GSE199360 | GEO | 2022/07/20

REPOSITORIES: GEO

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System-wide analyses reveal essential roles of N-terminal protein modification in bacterial physiology

Project description:Removal of the N-terminal formyl group on nascent proteins by peptide deformylase (PDF) is the most prevalent protein modification in bacteria that impacts over 90% of the proteome. PDF is essential and a critical target of antibiotic development; however, its role in bacterial physiology remains a long-standing question. In this work, we used system-wide and time-resolved analyses of the E. coli translatome and proteome to investigate the consequences of PDF inhibition at different stages. We found that PDF inhibition results in the rapid activation of cellular stress responses, especially those associated with defects in protein folding and perturbations at the bacterial inner membrane, followed by a global downregulation of metabolic pathways. Functional assays reveal the rapid hyperpolarization of the plasma membrane and impaired membrane integrity upon PDF inhibition, suggesting that disruption of plasma membrane is the most immediate and primary consequence of formyl group retention on nascent proteins. Our work resolves the physiological function of a ubiquitous N-terminal protein modification and uncovers its crucial role in maintaining the proper structure and function of the bacterial membrane.

2022-07-20 | PXD032725 | Pride

Proteome-wide analysis of the amino terminal status of Escherichia coli proteins at the steady-state and upon deformylation inhibition

Project description:A proteome-wide analysis was performed in Escherichia coli to identify the impact on protein N-termini of the antibiotic actinonin specifically inhibiting peptide deformylase (PDF). A new strategy and tool suite (SILProNaQ) was employed to provide large scale N-terminus acetylation yield quantitation. In control conditions, more than 1000 N-termini could be identified with 56 % Met removal, and additional modifications involving partial or complete N-acetylation (10%) and formyl retention (5%). Among the proteins undergoing these N-terminal modifications, some translocated membrane proteins were highlighted. The early time-course impact of actinonin was followed after the addition of bacteriostatic concentrations of the drug immediately slowing down the growth rate. Under these conditions, 25% of all proteins remain formylated after 10 min, a value reaching more than 60% of all characterized proteins after 40 min of treatment. The N-formylation rate on individual proteins increased with the same trend. Upon PDF inhibition, we finally show that two major categories of proteins retain their formyl group: a large number of inner membrane proteins and proteins involved in protein synthesis including many factors assisting the nascent chains in co-translational events.

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Proteome-wide analysis of the amino terminal status of Escherichia coli proteins at the steady-state

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Large scale investigation of the phage Vp16 PDF activity

Project description:Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this cotranslational mechanism. Recent genome sequencing and annotation studies highlighted over 2x104 putative peptide deformylase sequences. Furthermore, unpredicted modified bacterial PDF genes have been retrieved from many phages. Sequence comparisons with other known PDFs reveal that viral PDFs are devoid of the key ribosome-interacting C-terminal region. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity in vitro or in vivo. This investigation determines the activity of the recombinant Vp16 PDF in E. coli PDF defective cell to remove the N-formyl group at the protein N-terminus.

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Selective ribosome profiling reveals the co-translational chaperone action of trigger factor in vivo

Project description:As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting and folding factors. Here we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone Trigger Factor (TF) reveal that, while TF can interact with many polypeptides, Î²-barrel outer membrane proteins are the most prominent substrates. Loss of TF leads to broad outer membrane defects and premature, cotranslational protein translocation. While in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting ad folding factors. Examination of translation in the Gram-negative bacterium Escherichia coli, as well as an analysis of the interactions between nascent chains and the molecular chaperone Trigger Factor.

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Selective ribosome profiling reveals the co-translational chaperone action of trigger factor in vivo

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