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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

Whole lysate and ribosome enriched fractions analyses of the amino terminal status of Escherichia coli 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.

2015-07-21 | PXD001979 | Pride

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

2015-07-22 | PXD001983 | Pride

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

2015-07-21 | PXD002012 | Pride

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.

2017-09-25 | PXD006008 | Pride

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.

2011-12-09 | E-GEOD-33671 | biostudies-arrayexpress

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, β-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.

2011-12-09 | GSE33671 | GEO

Ribosome profiling reveals multiple roles of SecA in cotranslational protein export

Project description:SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also interacts with ribosomes. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA in vivo. SecA scans translating ribosomes at the plasma membrane and cotranslationally engages large periplasmic loops on inner membrane proteins. In coordination with the proton motive force, SecA resolves the cytoplasmic accumulation of periplasmic loops during cotranslational protein translocation. SecA also associates with a subset of secretory proteins at an early stage of translation and mediates their cotranslational transport, whereas the chaperone trigger factor (TF) delays SecA engagement on other secretory proteins to impose a posttranslational mode of translocation. The hydrophobicity of signal sequence is a determinant of nascent protein triage between SecA and TF. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria.

2022-05-16 | GSE185572 | GEO

Expression profiling of NM1 KO and WT mice

Project description:Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with the adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear function, we show that NM1 localizes preferentially to the plasma membrane. Deletion of NM1 causes more than 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrary, overexpression of NM1 in WT cells leads to additional 30% reduction of their survival. We have brought evidence that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. We used tissues from NM1 WT and KO mice with highest expression of NM1, lungs and heart (3 replicates each) and skin fibroblast derived from each mice (2 replicates each)

2015-12-05 | E-GEOD-75709 | biostudies-arrayexpress

Profiling antibody responses to M. tuberculosis proteins in TB suspects’ sera using full-proteome microarrays.

Project description:Understanding the immune response to tuberculosis requires greater knowledge of humoral responses. To characterize antibody targets and the effect of disease parameters on target recognition, we developed a systems immunology approach that integrated detection of antibodies against the entire Mycobacterium tuberculosis proteome, bacterial metabolic and regulatory pathway information, and patient data. Probing ~4,000 M. tuberculosis proteins with sera from >500 suspected tuberculosis patients worldwide revealed that antibody responses recognized ~10% of the bacterial proteome. This result defines the immunoproteome of M. tuberculosis, which is rich in membrane-associated and extracellular proteins. Most serum reactivity during active tuberculosis focused onto ~0.5% of the proteome. Within this pool, which is selectively enriched for extracellular proteins (but not for membrane-associated proteins), relative target preference varied among patients. The shift in relative M. tuberculosis protein reactivity observed with active tuberculosis defines the evolution of the humoral immune response during M. tuberculosis infection and disease.

2010-07-14 | GSE19433 | GEO

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