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Proteins are decorated with a diverse array of post-translational modifications (PTMs) that regulate their spatial and temporal functions. Recent mass spectrometry (MS)-based studies have identified hundreds of thousands of PTM sites in mammalian proteomes. However, the signaling cues and enzymes regulating individual sites are often not known and their functional roles remain uncharacterized. Quantification of PTM site stoichiometry can help in prioritizing sites for functional analyses and is important for constructing mechanistic models of PTM-dependent protein regulation. Here, we review the concept of PTM site stoichiometry, critically evaluate the merits and drawbacks of different MS-based methods used for quantifying PTM site stoichiometry, and discuss the usefulness and limitations of stoichiometry in informing on the biological function of modified sites.

Pyridoxal phosphate-dependent enzymes (PLP-DEs) are ubiquitous, catalytically diverse and essential for basic metabolism. A chemical proteomic strategy was developed for reporting on PLP-DEs in cells using functionalized PL-cofactor mimics. Biochemical, crystallographic and quantitative MS studies show that the probes are effectively absorbed, metabolized and integrated into biological systems. Our method enabled access to 73% of the Staphylococcus aureus PLP-ome and could be used to identify new PLP-DEs, screen for drug off-targets and probe enzyme active sites., Pyridoxalphosphat-abhängige Enzyme (PLP-DEs) sind vielfältig und für den Metabolismus essentiell. Zur Untersuchung zellulärer PLP-DEs wurde eine proteomische Methode mittels PL-Cofaktor Derivaten entwickelt. Durch biochemische und MS-basierte Studien konnte nachgewiesen werden, dass die Sonden effektiv aufgenommen, metabolisiert und in biologische Netzwerke eingebaut werden. Mithilfe dieser Strategie konnten 73% des PLP-oms von Staphylococcus aureus sowie neue PLP-DEs, off-targets von Medikamenten und aktive Zentren von Enzymen identifiziert werden.

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.

Pyridoxal phosphate-dependent enzymes (PLP-DEs) are ubiquitous, catalytically diverse and essential for basic metabolism. A chemical proteomic strategy was developed for reporting on PLP-DEs in cells using functionalized PL-cofactor mimics. Biochemical, crystallographic and quantitative MS studies show that the probes are effectively absorbed, metabolized and integrated into biological systems. Our method enabled access to 73% of the Staphylococcus aureus PLP-ome and could be used to identify new PLP-DEs, screen for drug off-targets and probe enzyme active sites., Pyridoxalphosphat-abhängige Enzyme (PLP-DEs) sind vielfältig und für den Metabolismus essentiell. Zur Untersuchung zellulärer PLP-DEs wurde eine proteomische Methode mittels PL-Cofaktor Derivaten entwickelt. Durch biochemische und MS-basierte Studien konnte nachgewiesen werden, dass die Sonden effektiv aufgenommen, metabolisiert und in biologische Netzwerke eingebaut werden. Mithilfe dieser Strategie konnten 73% des PLP-oms von Staphylococcus aureus sowie neue PLP-DEs, off-targets von Medikamenten und aktive Zentren von Enzymen identifiziert werden.

The biosynthesis of coenzyme A (CoA) from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide derivatives of pantothenate, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactive in vivo. In this study, we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with 50% inhibitory concentrations (IC50s) of ∼50 nM, and three others have submicromolar IC50s. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with the Plasmodium falciparum pantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA, and show that the compound inhibits the phosphorylation of [14C]pantothenate by the P. falciparum pantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase.