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Jessen, H.

Publications and source records attributed to Jessen, H..

17 recordsLinked to original sources
bioRxiv2026

The dynamic inositol phosphate network during development in Drosophila melanogaster

Inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are key regulators of cellular signaling and metabolism, yet developmental changes in the InsP/PP-InsP network in eukaryotic systems remain poorly understood. Here, we combine isomer-resolved capillary electrophoresis-mass spectrometry (CE-MS) with genetic perturbations to characterize InsP metabolism during the Drosophila melanogaster development. Our analyses reveal extensive developmental reprogramming of the InsP pathway, with early developmental stages exhibiting broad lower-order isomer diversity that progressively narrows into a more restricted metabolic state enriched in Ins(1,3,4,5,6)P5, InsP6, and PP-InsPs at later stages. Metamorphosis is characterized by a marked increase in 5-PP-InsP5, which emerges as the predominant pyrophosphate species in adults. Perturbation of dedicated kinases suggests that this metabolic remodeling is essential for developmental progression. RNAi-mediated IPK2 depletion disrupts higher-order InsP synthesis by reducing InsP5 levels, and results in failed metamorphosis. IPK1 depletion delays development and induces pronounced isomer-selective redistribution within the InsP5 pool, indicating network-level changes beyond simple precursor accumulation. IP6K depletion specifically abolishes 5-PP-InsP5 production, impairs tissue morphogenesis during pupal stages, and causes rapid post-eclosion lethality. Unexpectedly, depletion of either IPK2 or IP6K leads to the presence of 2-PP-InsP5. Collectively, these findings establish the Drosophila melanogaster InsP pathway as a dynamically regulated developmental network, particularly rich in previously undescribed InsP isomers, in which stage-specific metabolic remodeling parallels metamorphosis, tissue maturation, and adult viability.

biochemistry↗
bioRxiv2026

Inositol phosphates, pyrophosphates and the genes involved in their turnover in the streptophyte green alga Chara braunii

Inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are conserved signalling molecules, but their evolutionary origin and diversification in the green lineage remain poorly understood. Here we investigated the InsP network in the streptophyte alga Chara braunii, a key lineage lose to the origin of land plants. Using capillary electrophoresis-electrospray ionization mass spectrometry, we detected a broad spectrum of InsP and PP-InsP species from InsP3 to InsP8, including multiple positional isomers. Developmental profiling across dormant oospores, young thalli and mature thalli revealed extensive metabolic remodeling, with InsP6 as the dominant metabolite and distinct stage-dependent changes in lower InsPs and pyrophosphorylated species. Multiple PP-InsP5 and (PP)2-InsP4 isomers were identified, together with an unassigned additional InsP8-like signal, indicating further pathway complexity. Bioinformatic analyses identified candidate homologs of major InsP metabolic enzymes, supporting the presence of an enzymatic framework for InsP synthesis and turnover similar to land plants. Environmental perturbation revealed isomer-selective effects: prolonged light and dark phases strongly affected the accumulation of specific InsP5 and PP-InsP5 isomers, with 1-PP-InsP5 emerging as the most stimulus-responsive pyrophosphate species, whereas heat stress preferentially reduced 4-PP-InsP5. Together, these findings show that a structurally complex and environmentally responsive InsP network was already established in streptophyte algae before the emergence of land plants.

plant biology↗
bioRxiv2026

FRAPPe - a novel FRET-based biosensor to detect inorganic polyphosphates

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, is involved in a range of cellular processes, including phosphate storage, blood clotting, chromatin remodeling, RNA processing, and mitochondrial function. Despite its significance, tools for monitoring polyP with specificity and in real time are lacking, limiting insights into its dynamics. Here, we present FRAPPe (FRET-based Ratiometric Analysis of Polyphosphate), a FRET-based sensor for polyP. FRAPPe consists of an eCFP-eYFP FRET pair flanking CHAD, a polyP-binding domain. The sensor remains unresponsive to nucleic acids and other polyanions, while polyP binding induces a significant decrease in FRET, allowing quantitative estimation of polyP. We further show that FRAPPe can qualitatively estimate polyP levels from crude cell extracts, supporting its utility in high-throughput genetic and pharmacological screens to identify regulators of polyP metabolism and signaling.

biochemistry↗
bioRxiv2026

Inositol hexakisphosphate Functions as a Cofactor and Modulator of ADAR1 Activity

Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn{superscript 2}-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.

molecular biology↗
bioRxiv2025

Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP5-Binding Proteins in Plants

Inositol pyrophosphates (PP-InsPs) are central regulators of eukaryotic signaling events. While certain PP-InsP isomers have been conclusively linked to the regulation of phosphate homeostasis through interaction with SPX domain containing proteins in plants, the functions of the recently discovered isomer 4/6-PP-InsP5 remain largely unknown. Here, we employed two complementary affinity-based strategies - a matrix approach and a photoaffinity probe - to systematically identify 4/6-PP-InsP5-binding proteins in Arabidopsis thaliana. The two methods yielded partially overlapping protein sets, with photoaffinity enrichment likely capturing additional transient and/or weak interactions. Moreover, competition experiments with different isomers were applied to obtain information about potential isomer-specific interactions. As a proof-of-concept, one candidate interactor (FHA domain-containing protein AtFHA2) was shown to bind 4-PP-InsP5 in vitro with substantially higher affinity than InsP6. Thus, besides the SPX domain, FHA domain containing proteins, of which 18 exist in Arabidopsis, are potentially regulated by inositol pyrophosphates. More generally, our findings reveal a diverse protein network associated with 4/6-PP-InsP5 and establish a versatile platform for dissecting its biological roles in plants and other organisms.

biochemistry↗
bioRxiv2025

Mammalian mitochondrial polyphosphate is regulated by the 5-InsP7 synthase IP6K1

Inorganic polyphosphate (polyP) is a linear polymer of varying length composed of phosphate residues linked by phosphoanhydride bonds. PolyP remains poorly understood in mammals due to its low abundance and lack of information on its synthesis and regulation. Using synthetic polyP8 as a bait, we identified polyP interacting proteins involved in RNA splicing, transcription, and mitochondrial function. We developed a DAPI fluorescence-based assay to quantify the low levels of polyP present in mammalian cell lines and tissues, and detected a higher concentration of polyP in the mitochondria compared with the nucleus and post-mitochondrial fraction. We observed that mitochondrial polyP synthesis relies on active FoF1 ATP synthase and an intact proton gradient across the inner mitochondrial membrane. Our data shows that orthophosphate (Pi) is required for the generation of mitochondrial polyP and that the presence of ATP enhances Pi-driven polyP synthesis in isolated mitochondria. We discovered that the inositol pyrophosphate 5-InsP7, synthesized by IP6K1, regulates mitochondrial polyP levels. Mice and cells deficient in IP6K1 showed a significant reduction in mitochondrial polyP synthesis compared with wild type controls. Cells lacking IP6K1 also showed impaired mitochondrial respiration and membrane potential. The expression of active IP6K1, but not its catalytically inactive form, restored mitochondrial polyP synthesis and membrane potential in IP6K1 deficient cells, but mitochondrial respiration was rescued by expression of either active or inactive IP6K1. These data show that IP6K1 regulates mitochondrial function and polyP production through both the synthesis of 5-InsP7 and via a catalytic activity-independent mechanism. Our findings uncover a link between 5-InsP7, an energy sensor, and polyP, an energy store, in the regulation of mammalian mitochondrial homeostasis.

biochemistry↗
bioRxiv2025

Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol

Cells experience strong variations in the consumption and availability of inorganic phosphate (Pi). Since Pi is an essential macronutrient but excess Pi has negative impacts on nucleotide hydrolysis and metabolism, its concentration must be maintained in a suitable range. Conserved storage organelles, acidocalcisomes, provide this buffering function. We used acidocalcisome-like yeast vacuoles to study how such organelles are set up to for this task. Our combined in vitro and in vivo analyses revealed that their ATP-driven polyphosphate polymerase VTC converts cytosolic Pi into inorganic polyphosphates (polyP), which it transfers into the vacuole lumen. Luminal polyphosphatases immediately hydrolyse this polyP to establish a growing reservoir of vacuolar Pi. Product inhibition by this Pi pool silences the polyphosphatases, caps Pi accumulation, and favours vacuolar polyP storage. Upon cytosolic Pi scarcity, the declining inositol pyrophosphate levels activate the vacuolar Pi exporter Pho91 to replenish cytosolic Pi. In this way, acidocalcisome-like vacuoles constitute a feedback-regulated buffering system for cytosolic Pi, which the cells can switch between Pi accumulation, Pi release, and high-capacity phosphate storage through polyP.

cell biology↗
bioRxiv2025

The small membrane protein YohP induces membrane depolarization and ppGpp accumulation in Escherichia coli.

Small membrane proteins represent an abundant and ubiquitous class of proteins that are often up-regulated when cells encounter unfavorable conditions, yet details about their exact function are largely missing. In bacteria, these proteins consist of typically less than 50 amino acids and contain a single transmembrane domain, but lack any detectable catalytic activity. Thus, the benefit of producing these proteins during stress conditions is unknown. In the current study we used a multidisciplinary approach to determine the function of the 27 amino acid long protein YohP in E. coli. Our proteomics approach revealed that YohP production leads to an up-regulation of proteins involved in membrane protection and to a down-regulation of many enzymes involved in key metabolic processes, such as nucleotide biosynthesis. Further biochemical characterizations revealed increased cardiolipin content in the membrane, a partial dissipation of the membrane potential and reduced membrane fluidity in YohP-containing membranes. Finally, our data show that YohP production induces the stringent response and leads to elevated levels of (p)ppGpp. Overall, our data indicate that the YohP-induced proteome and membrane changes initiate a state of metabolic silencing that protects E. coli against stress and helps to conserve cellular resources.

microbiology↗
bioRxiv2025

KIDINS220 and InsP8 safeguard the stepwise regulation of phosphate exporter XPR1

XPR1 is emerging as the only known inorganic phosphate (Pi) exporter in humans, critical for Pi homeostasis, with its activity stimulated by inositol pyrophosphate InsP8 and regulated by neuronal scaffold protein KIDINS220. Our structural studies reveal InsP8 specifically activates XPR1 in a stepwise manner, involving profound SPX domain movements. Each XPR1 subunit functions with four gating states, in which Pi permeates a constriction site via a "knock-kiss-kick" process. In contrast, KIDINS220 delicately stabilizes XPR1 in a closed conformation through multiple mechanisms, one of which involves trapping the XPR1 1 helix--critical for InsP8 binding--within an interaction hub. InsP8 serves as a key to release KIDINS220s restraint, reinforcing a "key-to-locks" mechanism to safeguard the stepwise activation. Additionally, our study provides direct structural insights into XPR1-associated neuronal disorders and highlights the evolutionary conservation and divergence among XPR1 orthologues, offering a comprehensive understanding of Pi homeostasis across species.

molecular biology↗
bioRxiv2024

Structural Insights into Broad-Range Polyphosphate Kinase 2 II Enzymes Applicable for Pyrimidine Nucleoside Diphosphate Synthesis

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP-consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85% of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49%. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

biochemistry↗
bioRxiv2024

The wheat VIH2-3B, a functional PPIP5K controls the localization of fasciclin-like arabinogalactan protein

Inositol pyrophosphates (PP-InsPs) are important signalling molecules that participate in multiple physiological processes across a wide range of eukaryotes. Metabolic pathway kinases (VIP1/VIHs) leading to the production of PP-InsPs are now well characterized in yeast and plants. Previously, the wheat (Triticum aestivum L.) inositol pyrophosphate kinase (TaVIH2) was shown to encode a catalytic active kinase domain. Heterologous expression of TaVIH2 in Arabidopsis thaliana was shown to enhance drought tolerance by modulating the cell composition. In this study, we attempted to identify the interacting protein targets of wheat VIH2-3B using a yeast two-hybrid (Y2H) cDNA library screen, which led to the identification of 52 putative interactors that are primarily involved in cell wall-related functions. Notably, fasciclin-like arabinogalactan protein (FLA7), a glycosylphosphatidyl inositol (GPI)-anchored protein, emerged as the most frequently interacting partner. Further analysis using pulldown assays validated the interaction between TaVIH2-3B and TaFLA7 in vivo. Using the reporter fusion studies, we observed the localization of TaFLA7 to be a plasma membrane and this localization of the TaFLA7 was perturbed in the yeast vip1{Delta} strain. The expression of TaVIH2-3B bearing PPIP5K enzymatic activity in yeast mutants rescued the level of IP8 and restore the localisation of the TaFLA7 to the membrane. Expression analysis of TaFLA7 revealed a differential expression response to drought in wheat shoot tissues. TaFLA7 was also found to be highly expressed during grain development, particularly in the endosperm and seed coat during grain maturation. Taken together, these findings highlight the potential role of TaVIH2 in cell wall remodelling and stress response pathways, offering new insights into the functional roles of VIH proteins in plants.

plant biology↗
bioRxiv2024

Inositol Hexaphosphate (InsP6) Activates the HDAC1/3 Epigenetic Axis toMaintain Intestinal Barrier Function

While HDAC inhibition shows promise in cancer treatment, pan-HDAC inhibitors cause gastrointestinal issues in 48% of patients. Understanding HDAC activation mechanisms is crucial to treat diverse diseases beyond cancer. Our study reveals the essential role of inositol polyphosphate multikinase (IPMK) and inositol hexakisphosphate (InsP6 or phytic acid), enriched in vegan diets, in activating the HDAC3 epigenetic axis and maintaining intestinal barrier integrity. IPMK binds to HDAC3, driving InsP6 synthesis, which selectively activates HDAC3 at 10nM concentration by recruiting the DAD domain of its corepressor protein. IPMK deletion diminishes HDAC3 activation, leading to histone hyperacetylation and MMP gene transcription, compromising intestinal barrier integrity. InsP6 treatment is sufficient to rescue these effects. In inflammatory bowel disease, diminished IPMK levels exacerbated intestinal permeability, while oral InsP6 treatment mitigated gut permeability by restoring the HDAC3 epigenetic axis, indicating the clinical implications of the IPMK-HDAC3 epigenetic axis and therapeutic potential of phytic acid.

cell biology↗
bioRxiv2024

Pools of independently cycling inositol phosphates revealed by pulse labeling with 18O-water

Inositol phosphates control many central processes in eukaryotic cells, including nutrient availability, growth, and motility. Kinetic resolution of a key modulator of their signaling functions, the turnover of the phosphate groups on the inositol ring, has been hampered by slow uptake, high dilution, and constraining growth conditions in radioactive pulse-labeling approaches. Here, we demonstrate rapid (seconds to minutes), non-radioactive labeling of inositol polyphosphates through 18O-water in yeast, amoeba and human cells, which can be applied in any media. In combination with capillary electrophoresis and mass spectrometry, 18O-water labeling simultaneously dissects the in vivo phosphate group dynamics of a broad spectrum of even rare inositol phosphates. The improved temporal resolution allowed us to discover vigorous phosphate group exchanges in some inositol poly- and pyrophosphates, whereas others remain remarkably inert. Our observations support a model in which the biosynthetic pathway of inositol poly- and pyrophosphates is organized in distinct, kinetically separated pools. While transfer of compounds between those pools is slow, each pool undergoes rapid internal phosphate cycling. This might enable the pools to perform distinct signaling functions while being metabolically connected.

biochemistry↗
bioRxiv2022

Biomimetic S-adenosylmethionine regeneration for nucleophilic and radical alkylation reactions and aminopropyltransfer

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report an efficient one-pot system to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

biochemistry↗
bioRxiv2022

Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly-created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references, and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in Nature. Analysis of feces and the commercial mouse diet suggest the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.

biochemistry↗
bioRxiv2022

Stable isotopomers of myo-inositol to uncover the complex MINPP1-dependent inositol phosphate network

The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers central to a myriad of cellular processes. However, we have an incomplete understanding of InsP metabolism because the available analytical toolset for inositol phosphates is rather limited. Here, we have synthesized and utilized fully and unsymmetrically 13C-labeled myo-inositol and inositol phosphates. These probes were applied in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to further annotate central aspects of InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR - down to individual enantiomers - which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme, that contributes to cellular homeostasis. Full characterization of MINPP1 activity in vitro and in cells, provided a clear picture of this multifunctional phosphatase. Metabolic labeling with stable isotopomers thus constitutes a powerful tool for investigating InsP networks in a variety of different biological contexts.

biochemistry↗