Biology subjects

Jessen, H. J.

Publications and source records attributed to Jessen, H. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Fasting disrupts the InsP6 HDAC3 axis to drive ER stress-mediated clearance of DNA-damaged cells and enforce tissue quality control.

Fasting drives metabolic adaptation but also elicits acute cellular stress. How this stress shapes tissue integrity is unknown. Here, we show that in the intestine, fasting depletes growth factor signaling, which triggers cellular stress. This response functions as a tissue quality-control checkpoint that selectively eliminates pre-existing DNA-damaged cells while sparing healthy counterparts. A short-term fast diminishes TGF-{beta} signaling and elicits endoplasmic reticulum (ER) stress, driving DNA-damaged intestinal cells beyond an apoptotic threshold, thereby reducing the inflammatory burden. Mechanistically, loss of TGF-{beta} signaling triggers FBXO22-Cullin1-mediated degradation of the inositol kinase IPMK, leading to depletion of inositol hexaphosphate (InsP). InsP loss attenuates HDAC3 activity and initiates coordinated epigenetic and post-translational reprogramming, thereby increasing CDK5RAP3 abundance. Elevated CDK5RAP3 inhibits ribosomal RPL26 UFMylation, thereby amplifying ER stress and selectively licensing apoptosis in DNA-damaged cells. Collectively, fasting disrupts a TGF-{beta}-InsP6-HDAC3 axis to drive ER stress-dependent clearance of DNA-damaged cells, enforcing tissue quality control.

cell biology↗

bioRxiv2026

Simultaneous Capillary Electrophoresis - Mass Spectrometry profiling of (p)ppGpp and dinucleoside polyphosphates reveals oscillatory (p)ppGpp dynamics and a potential synchronisation between alarmones

To survive stressful conditions, bacteria enter the stringent response mediated by the magic spot nucleotides (MSN) guanosine 35-bispyrophosphate (ppGpp) and guanosine 3-diphosphate 5-triphosphate (pppGpp). Another group of so-called alarmones elevated under stress are dinucleoside polyphosphates (NpnNs), such as diadenosine triphosphate (Ap3A). How these two groups of alarmones intersect with one another is poorly understood. Here, we present a sensitive method using capillary electrophoresis coupled to mass spectrometry (CE-MS) with heavy isotope labeled internal references to separate and quantify magic spot nucleotides in parallel with NpnNs. This approach uncovered oscillatory fluctuations in MSN and some NpnN during exponential growth, suggesting more dynamic regulation of the stringent response than previously thought. In addition, up to eleven NpnNs were detected after prolonged growth. NpnN levels were also quantified under various amino acid starvation conditions, revealing a pronounced accumulation in late stationary phase. In summary, our parallel assignment and quantification of highly charged signaling molecules in bacteria under stress conditions paves the way to better study and understand the cross-talk between different alarmones.

biochemistry↗

bioRxiv2025

Controlled Delivery and Light-Induced Release of Magic Spot Nucleotides in Escherichia coli

The "magic spot nucleotides" (MSNs) ppGpp and pppGpp (also: (p)ppGpp) are bacterial alarmones central to the conserved stringent response, a stress adaptation mechanism that helps bacteria adapt to stress conditions and hostile environments. Current strategies to manipulate MSN levels rely mainly on genetic or environmental approaches, which are slow and lack temporal control. Chemical tools such as photocaged MSN analogues could provide such temporal control over MSN levels. However, the high negative charge of MSNs prevents spontaneous passage through the complex bacterial cell envelope. Here, we report the synthesis of photocaged, clickable, and isotope-labeled MSN analogues and their delivery into Escherichia coli comparing different approaches. A cyclodextrin-based synthetic nucleotide transporter provides particular advantages. Upon 400 nm irradiation, these probes were photo-released inside living cells, where we tracked their conversion from pppGpp to ppGpp by capillary electrophoresis mass spectrometry and studied their ability to alter growth in a (p)ppGpp0 mutant. This work provides the first demonstration that highly charged, photocaged MSNs can traverse the bacterial envelope, be photo-released intracellularly, and be metabolically tracked in real time. These probes lay the foundation for spatially and temporally controlled studies of MSN function and of other highly negatively charged metabolites in bacteria.

biochemistry↗

bioRxiv2025

A Screening Approach Unveils an Unknown Mn2+ - dependent Endopolyphosphatase Activity in Yeast

Inorganic polyphosphate (polyP) is a ubiquitous biopolymer composed of multiple orthophosphates connected by energy-rich phosphoanhydride bonds. In organisms, polyP is digested by two types of enzymes: exopolyphosphatases, which shorten the chain from the ends by cleaving off monophosphate units, and endopolyphosphatases, which cut the chain internally. While several continuous methods are available to monitor exopolyphosphatase activity, endopolyphosphatase activity assays are less common and typically involve multiple tedious steps. Here, we introduce FRET-polyP8, a novel probe for real-time detection of endopolyphosphatase activity. The FRET assay enabled rapid, highly sensitive, single-step detection of specific endopolyphosphatase activity both from isolated proteins and cell extracts. The simple read-out additionally enabled enzyme inhibitor screening. Furthermore, a novel Mn2+-dependent endopolyphosphatase activity in bakers yeast was detected in a quadruple mutant, highlighting the ability to screen for metal-dependence of new endopolyphosphatase activity. This approach thus represents a significant addition to existing methodologies, facilitating the discovery and classification of new endopoly-phosphatases and their inhibitors to advance our understanding of polyP metabolism and regulation.

biochemistry↗

bioRxiv2024

Lotus japonicus VIH2 is an inositol pyrophosphate synthase that regulates arbuscular mycorrhiza

Plant yield is often maximized by the extensive use of mineral fertilizers, which, however, has severe environmental consequences. Phosphate is particularly problematic, as it represents a globally limited resource, and its runoff and soil erosion threaten open water bodies. Many crops engage in arbuscular mycorrhizal (AM) symbiosis with nutrient-acquiring fungi, aiding in the uptake of phosphate and other mineral nutrients. However, AM colonization is strongly reduced under high soil phosphate levels. A mechanistic understanding of phosphate sensing, phosphate starvation responses, and their connection to AM remains enigmatic. Here, we show that in Lotus japonicus, low-abundant, energy-rich inositol pyrophosphates act as master regulators of AM, orchestrating the crosstalk between phosphate starvation responses and plant root endosymbiosis. These findings hold promise for breeding nutrient-efficient crops.

plant biology↗

bioRxiv2024

Association of the benzoxazinoid pathway with boron homeostasis in maize

Both deficiency and toxicity of the micronutrient boron lead to severe reductions in crop yield. Despite this agricultural importance, the molecular basis underlying boron homeostasis in plants is not resolved. To identify molecular players involved in boron homeostasis in maize (Zea mays), we measured boron levels in the Goodman-Buckler association panel and performed genome-wide association studies. These analyses detected the benzoxazinless (bx) gene, bx3, involved in the biosynthesis of benzoxazinoids, like DIMBOA, major defense compounds in maize. Genes involved in DIMBOA biosynthesis are all located in close proximity in the genome and the biosynthesis mutants, including bx3 are all DIMBOA deficient. We show that the bx3 mutant has enhanced boron concentration in leaves compared to the B73 control plants, which correlates with enhanced leaf tip necrosis, a phenotype associated with boron toxicity. In contrast, other DIMBOA-deficient maize mutants did not show altered boron levels nor the leaf tip necrosis phenotype, suggesting that boron is not associated with DIMBOA. Instead, our analyses suggest that the accumulation of boron is linked to the benzoxazinoid intermediates indolin-2-one (ION) and 3-hydroxy-ION. Therefore, our results connect boron homeostasis to the benzoxazinoid plant defense pathway through bx3 and specific intermediates, rendering the benzoxazinoid biosynthesis pathway a potential target for crop improvement in inadequate boron conditions. One sentence summaryGWAS identified benzoxazinless3 in the benzoxazinoid biosynthesis pathway to play a role in boron homeostasis in maize.

plant biology↗

bioRxiv2024

NUDIX Hydrolases Target Specific Inositol Pyrophosphates and Regulate Phosphate and Iron Homeostasis, and the Expression of Defense Genes in Arabidopsis

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher-order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange (SAX)-HPLC, PAGE, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer-selective and Mg2+-dependent. Specifically, subclade I NUDTs preferentially hydrolyze 4-InsP7, while subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher-order mutants of subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wildtype (WT) and subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we found that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

plant biology↗

bioRxiv2024

Transport and InsP8 activation mechanisms of the human inorganic phosphate exporter XPR1

Inorganic phosphate (Pi) has essential metabolic and structural roles in living organisms. The Pi exporter, XPR1/SLC53A1, is critical for maintaining cellular Pi homeostasis. When intercellular Pi is high, cells synthesize inositol pyrophosphate (1,5-InsP8) - a signaling molecule that is required for XPR1 function. Inactivating mutations of XPR1 lead to brain calcifications causing neurological symptoms that include migraine, movements disorders, psychosis, and dementia. Distinct cryo-electron microscopy structures of dimeric XPR1 and functional characterization define the substrate translocation pathway and delineate how binding of InsP8 initiates the transport cycle. InsP8 binding rigidifies the intracellular SPX domains with InsP8 acting as a bridge between dimers and the SPX and transmembrane domains. When locked in this state, the C-terminal tail is sequestered revealing the entrance to the transport pathway, thus explaining the obligate roles of the SPX domain and InsP8. Together, these findings advance our understanding of XPR1 transport activity and expand opportunities for rationalizing disease mechanisms and therapeutic intervention.

biophysics↗

bioRxiv2024

Inositol pyrophosphate catabolism by three families of phosphatases controls plant growth and development

Inositol pyrophosphates (PP-InsPs) are nutrient messengers whose cellular concentration must be tightly regulated. Diphosphoinositol pentakisphosphate kinases (PPIP5Ks) generate the active signaling molecule 1,5-InsP8. PPIP5Ks contain additional phosphatase domains involved in PP-InsP catabolism. Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSPs) and NUDIX phosphatases (NUDTs) also hydrolyze PP-InsPs. Here we dissect the relative contributions of the three different phosphatase families to plant PP-InsP catabolism and nutrient signaling. We report the biochemical characterization of inositol pyrophosphate phosphatases from Arabidopsis and Marchantia polymorpha. Overexpression of different PFA-DSP and NUDT enzymes affects PP-InsP levels and leads to stunted growth phenotypes in Arabidopsis. nudt17/18/21 knock-out mutants have altered PP-InsP pools and gene expression patterns, but no apparent growth defects. In contrast, Marchantia polymorpha Mppfa-dsp1ge, Mpnudt1ge and Mpvip1ge mutants display severe growth and developmental phenotypes associated with changes in cellular PP-InsP levels. Analysis of Mppfa-dsp1geand Mpvip1ge supports a role for PP-InsPs in Marchantia phosphate signaling, and additional functions in nitrate homeostasis and cell wall biogenesis. Simultaneous removal of two phosphatase activities enhances the observed growth phenotypes. Taken together, PPIP5K, PFA-DSP and NUDT inositol pyrophosphate phosphatases play important roles in growth and development by collectively shaping plant PP-InsP pools. Author summaryOrganisms must maintain adequate levels of nutrients in their cells and tissues. One such nutrient is phosphorus, an essential building block of cell membranes, nucleic acids and energy metabolites. Plants take up phosphorus in the form of inorganic phosphate and require sufficient cellular phosphate levels to support their growth and development. It has been shown that plants and other eukaryotic organisms "measure" cellular phosphate levels using inositol pyrophosphate signaling molecules. The concentration of inositol pyrophosphates serves as a proxy for the cellular concentration of inorganic phosphate, and therefore inositol pyrophosphate synthesis and degradation must be tightly regulated. Here, we report that three different families of enzymes contribute to the degradation of inositol pyrophosphates in plants. The different phosphatases together shape cellular inositol pyrophosphate pools and thereby affect inorganic phosphate levels. Loss-of-function mutants of the different enzymes display additional defects in nitrate levels and cell wall architecture, suggesting that inositol pyrophosphates regulate cellular processes beyond inorganic phosphate homeostasis.

plant biology↗

bioRxiv2023

Conservation of heat stress acclimation by the inositol polyphosphate multikinase, IPMK responsible for 4/6-InsP7 production in land plants

Inositol pyrophosphates (PP-InsPs) are soluble cellular messengers that integrate environmental cues to induce adaptive responses in eukaryotes. In plants, the biological functions of various PP-InsP species are poorly understood, largely due to the absence of canonical enzymes present in other eukaryotes. The recent identification of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP7 in the eudicot Arabidopsis thaliana, further highlights the intricate PP-InsP signalling network employed by plants. The abundance of 4/6-InsP7 in land plants, the enzyme(s) responsible for its synthesis, and the physiological functions of this species are all currently unknown. In this study, we show that 4/6-InsP7 is the major PP-InsP species present across land plants. Our findings demonstrate that the Arabidopsis inositol polyphosphate multikinase (IPMK) homolog, AtIPK2 generates 4/6-InsP7 in vitro. Furthermore, the cellular level of 4/6-InsP7 is controlled by the two Arabidopsis IPMK isoforms, AtIPK2 and AtIPK2{beta}. Notably, the activity of these IPMK proteins is critical for heat stress acclimation in Arabidopsis. During heat stress, the expression of genes encoding various heat shock proteins controlled by the heat shock factors (HSFs) is affected in the AtIPK2-deficient plants. Furthermore, we show that the transcription activity of HSF is regulated by the AtIPK2 proteins. Our parallel investigations using the liverwort Marchantia polymorpha suggest that the InsP6 kinase activity of IPMK and the role of IPMK in regulating the heat stress response are evolutionarily conserved. Collectively, our study indicates that IPMK has played a critical role in transducing environmental cues for biological processes during land plant evolution.

plant biology↗

bioRxiv2023

PenTag, a Versatile Platform for Covalent Bioconjugation, Purification, and Tagging of Proteins Towards the Development of Novel Biohybrid Material Systems

The site-specific and covalent conjugation of proteins on solid supports and in hydrogels is the basis for the synthesis of biohybrid materials offering broad applications. Current methods for conjugating proteins to desired targets are often challenging due to unspecific binding, unstable (non-covalent) coupling, or expensive and difficult-to-synthesize ligand molecules. Here, we present PenTag, an approach for the biorthogonal, highly-specific and covalent conjugation of a protein to its ligand for various applications in materials sciences. We engineered penicillin- binding protein 3 (PBP3) and showed that this protein can be used for the stable and spontaneous conjugation of proteins to dyes, polymers, or solid supports. We applied PenTag as a crosslinking tool for synthesizing stimuli-responsive hydrogels or for the development of a biohybrid material system performing computational operations emulating a 4:2 encoder. Based on this broad applicability and the use of a small, cheap and easy-to-functionalize ligand and a stable, soluble recombinant protein, we see PenTag as a versatile approach towards biohybrid material synthesis.

synthetic biology↗

bioRxiv2023

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts Through Specific RelA/SpoT Homologs

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 in Arabidopsis 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 in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. 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 of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. A. thaliana mutants 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.

biochemistry↗

bioRxiv2023

Inositol pyrophosphate dynamics in yeast reveals control of the PHO starvation program through 1,5-IP8 and the SPX domain of the CDK inhibitor Pho81

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates (IPPs) and SPX receptor domains. Since cells synthesize various IPPs and SPX domains bind them promiscuously, it is unclear whether a specific IPP regulates SPX domains in vivo, or whether multiple IPPs act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the IPP 1-IP7, we now show that the levels of all detectable IPPs of yeast, 1-IP7, 5-IP7 and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses. Significance statementCytosolic Pi is of prime importance for cellular bioenergetics because Pi influences free energy of nucleotide hydrolysis and the metabolite fluxes through glycolysis and oxidative phosphorylation. Eukaryotic cells use the INPHORS pathway to signal Pi via SPX domains and their ligands, inositol pyrophosphates (IP7, IP8), which control Pi homeostasis through a network of target proteins that import, export, store or detoxify Pi. Studies with different systems failed to yield a coherent model on this regulation. We performed the first time-resolved profiling of the full isomer spectrum of inositol pyrophosphates in yeast and dissected the isomer that is relevant to intracellular Pi signaling. Our results can be combined with existing observations from plants, mammals, and other fungi to support a unified model of Pi signaling across all eukaryotic kingdoms, which is in accord with the fundamental importance of Pi management for metabolism.

cell biology↗

bioRxiv2022

β-Lapachone Regulates Mammalian Inositol Pyrophosphate Levels in an NQO1- and Oxygen-dependent Manner

1Inositol 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 {beta}-lapachone ({beta}-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that {beta}-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 {beta}-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 {beta}-lap. The data presented here unveil new aspects of {beta}-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. 2 Significance 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 {beta}-lapachone ({beta}-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, {beta}-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 {beta}-lap efficacy in a combination chemotherapy against pancreatic cancer.

biochemistry↗

bioRxiv2022

Metabolic consequences of polyphosphate synthesis and imminent phosphate limitation

Cells stabilize intracellular inorganic phosphate (Pi) to compromise between large biosynthetic needs and detrimental bioenergetic effects of Pi. Pi homeostasis in eukaryotes employs SPXs domains, which are receptors for inositol pyrophosphates. We explored how polymerization and storage of Pi in acidocalcisome-like vacuoles supports S. cerevisiae metabolism and how these cells recognize Pi scarcity. Whereas Pi starvation affects numerous metabolic pathways, beginning Pi scarcity affects few metabolites. These include inositol pyrophosphates and ATP, a low-affinity substrate for inositol pyrophosphate-synthesizing kinases. Declining ATP and inositol pyrophosphates may thus be indicators of impending Pi limitation. Actual Pi starvation triggers accumulation of the purine synthesis intermediate 5- aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates Pi-dependent transcription factors. Cells lacking polyphosphate show Pi starvation features already under Pi-replete conditions, suggesting that vacuolar polyphosphate supplies Pi for metabolism even when Pi is abundant. However, polyphosphate deficiency also generates unique metabolic changes that are not observed in starving wildtype cells. Polyphosphate in acidocalcisome-like vacuoles may hence be more than a global phosphate reserve and channel Pi to preferred cellular processes. Abstract importanceCells must strike a delicate balance between the high demand of inorganic phosphate (Pi) for synthesizing nucleic acids and phospholipids, and its detrimental bioenergetic effects by reducing the free energy of nucleotide hydrolysis. The latter may stall metabolism. Therefore, microorganisms manage the import and export of phosphate, its conversion into osmotically inactive inorganic polyphosphates, and their storage in dedicated organelles, acidocalcisomes. Here, we provide novel insights into metabolic changes that cells may use to signal declining phosphate availability in the cytosol and differentiate it from actual phosphate starvation. We also analyze the role of acidocalcisome-like organelles in phosphate homeostasis. This uncovers an unexpected role of the polyphosphate pool in these organelles under phosphate-rich conditions, indicating that its metabolic roles go beyond that of a phosphate reserve for surviving starvation.

cell biology↗

bioRxiv2022

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in enteric nervous system

Inositol pyrophosphates (PP-IPs) regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled PP-IP levels in mammalian organs using a novel HILIC-MS/MS protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of IP7 and its precursor IP6. Although their absolute levels in the GIT is diet-dependent, elevated IP7 metabolism still exists under dietary regimes devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. Additionally, HILIC-MS/MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition induced the gene sets associated with mature neurons such as inhibitory, GABAergic and dopaminergic neurons, concomitantly with suppression of those for neural progenitor/stem cells and glial cells. In addition, IP6K2 inhibition explicitly affected transcript levels of certain genes modulating neuronal differentiation and functioning, implying critical roles of IP6K2-IP7 axis in developmental and functional regulation of enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7--a highly active IP6K2-IP7 pathway is conducive to enteric nervous system.

molecular biology↗

bioRxiv2022

Arabidopsis PFA-DSP-type phosphohydrolases target specific inositol pyrophosphate messengers

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologs display phosphohydrolase activity with a high specificity for the 5-{beta}-phosphate of inositol pyrophosphates. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin-sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-{beta}-phosphate specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

plant biology↗

bioRxiv2021

Photoaffinity capture compounds to profile the Magic Spot Nucleotide interactomes

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. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/472736v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@15cfc02org.highwire.dtl.DTLVardef@1a4ec1borg.highwire.dtl.DTLVardef@88fd27org.highwire.dtl.DTLVardef@1a42248_HPS_FORMAT_FIGEXP M_FIG Molecular fishing: a family of trifunctional photoaffinity capture compounds enables the identification of Magic Spot Nucleotide receptors by a chemoproteomics approach. C_FIG

biochemistry↗