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1AbstractInositol pyrophosphates (PP-InsPs) are energetic signalling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involves PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signalling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly β-lapachone (β-lap), under normoxic and hypoxic conditions enabled us to produce ROSin celluloand to show that β-lap treatment caused PP-InsP changes that are oxygen dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that β-lap requires NQO1-bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with β-lap. The data presented here unveil new aspects of β-lap pharmacology and its impact on PP-InsP levels. Our identification of different quinone drugs as modulators of PP-InsP synthesis will allow to better appreciate their overall impact on cellular function.2Significance StatementInositol pyrophosphates (PP-InsPs) are messenger molecules regulating various functions in mammals. They are associated with the oxidative stress response, but the underlying mechanism is unclear. We investigate PP-InsP signalling in mammalian cells subjected to reactive oxygen species (ROS). Applying the quinone β-lapachone (β-lap) generated intracellular ROS resulting in decreased PP-InsP levels. This indicates a key role of PP-InsPs in cellular signalling under oxidative stress. Moreover, β-lap mediated PP-InsP signalling required oxygen and the enzyme NAD(P)H:quinone oxidoreductase-1 (NQO1). Since quinone drugs are cytotoxic, our data provide a basis for further investigations into the role of PP-InsPs during quinone-dependent chemotherapies. This is of special relevance since a phase II clinical trial demonstrated β-lap efficacy in a combination chemotherapy against pancreatic cancer.

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival when confronted with adverse external challenges. In times of antibiotic crisis, a detailed understanding of the stringent response is of critical importance, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium cell lysates applying a family of trifunctional photoaffinity capture compounds. We introduce different MSN probes covering diverse phosphorylation patterns, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of diverse putative MSN receptors both from cytosolic and membrane fractions that, upon validation, unveil new MSN targets. We find, for example, that the dinucleoside polyphosphate hydrolase activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The photoaffinity capture compounds described herein will be useful to identify MSN interactomes under varying conditions and across bacterial species.TOCMolecular fishing: a family of trifunctional photoaffinity capture compounds enables the identification of Magic Spot Nucleotide receptors by a chemoproteomics approach.

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RSH enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity inArabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels inA. thalianaupon infection with the pathogenPseudomonas syringaepv.tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation ofRSH2upon flg22 treatment and bothRSH2andRSH3afterPstDC3000infection.A. thalianamutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting involvement of these synthases in PAMP-triggered innate immunity responses to pathogens within the chloroplast.