N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Myristoylation often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence suggests additional regulatory roles for myristoylation on proteins. Here we describe the myristoylated proteome of Toxoplasma gondii using chemoproteomic methods and show that a small-molecule NMT inhibitor developed against related Plasmodium spp. is also functional in Toxoplasma. We identify myristoylation on a transmembrane protein, the microneme protein 7 (MIC7), which enters the secretory pathway in an unconventional fashion with the myristoylated N-terminus facing the lumen of the micronemes. MIC7 and its myristoylation play a crucial role in the initial steps of invasion, likely during the interaction with and penetration of the host cell. Myristoylation of secreted eukaryotic proteins represents a substantial expansion of the functional repertoire of this co-translational modification.
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 5, 6 and 7. Source data for mass spectrometry proteomics results can be found in Supplementary files 1-4. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD019677.
Global profiling of myristoylation in Toxoplasma gondii.ProteomeXchange, PXD019677.
Differential protein phosphorylation during stage conversion in Toxoplasma gondiiProteomeXchange, PXD019729.
- Malgorzata Broncel
- Caia Dominicus
- Stephanie D Nofal
- Alex Hunt
- Bethan Alexandra Wallbank
- Joanna Claire Young
- Moritz Treeck
- Malgorzata Broncel
- Caia Dominicus
- Moritz Treeck
- Stephen Matthews
- Edward J Bartlett
- Edward W Tate
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Dominique Soldati-Favre, University of Geneva, Switzerland
© 2020, Broncel et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Genes of unknown function are among the biggest challenges in molecular biology, especially in microbial systems, where 40–60% of the predicted genes are unknown. Despite previous attempts, systematic approaches to include the unknown fraction into analytical workflows are still lacking. Here, we present a conceptual framework, its translation into the computational workflow AGNOSTOS and a demonstration on how we can bridge the known-unknown gap in genomes and metagenomes. By analyzing 415,971,742 genes predicted from 1749 metagenomes and 28,941 bacterial and archaeal genomes, we quantify the extent of the unknown fraction, its diversity, and its relevance across multiple organisms and environments. The unknown sequence space is exceptionally diverse, phylogenetically more conserved than the known fraction and predominantly taxonomically restricted at the species level. From the 71 M genes identified to be of unknown function, we compiled a collection of 283,874 lineage-specific genes of unknown function for Cand. Patescibacteria (also known as Candidate Phyla Radiation, CPR), which provides a significant resource to expand our understanding of their unusual biology. Finally, by identifying a target gene of unknown function for antibiotic resistance, we demonstrate how we can enable the generation of hypotheses that can be used to augment experimental data.
Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.