Identification of a distinct family of polyphosphate kinases relevant to energy homeostasis in Crenarchaeota.

Inorganic polyphosphate is highly conserved, critical, yet poorly understood polymer that regulates diverse cellular functions in mammals. Its importance is well established in coagulation, inflammation, mitochondrial function, and stress responses, though the molecular mechanisms for these effects remain only partly understood. Fundamental questions also persist regarding its physiological concentration, chain-length distributions, and the mechanisms that regulate its behavior in specific cellular compartments. Progress is limited by the absence of a known mammalian polyphosphate-synthesizing enzyme. Despite this, recent studies have broadened the scope of polyphosphate biology, suggesting roles in protein phase separation, ATP-independent chaperone activity, metabolic regulation, and intracellular signaling. Polyphosphate modulates the mitochondrial permeability transition pore through calcium-dependent regulation and activates factor XII in coagulation. Findings have also introduced potential connections between polyphosphate and processes such as neurodegeneration, cancer, and tissue regeneration. Despite this expanding landscape, many biological effects remain difficult to interpret due to incomplete mapping of protein targets and longstanding technical limitations in detecting and quantifying polyP. This review integrates molecular protein-interaction mechanisms with compartment-specific functions and disease physiology, providing a clearer mechanistic framework while identifying key conceptual and methodological gaps and outlining priorities for advancing polyphosphate research in mammalian systems.

Discovery of polyphosphate-interacting human lysine-rich proteins and their functional implications.

Chemorepulsion mechanisms for eukaryotic cells are poorly understood. We performed proteomics and phosphoproteomics to elucidate how Dictyostelium discoideum responds to its two endogenous chemorepellent signals, the protein AprA and inorganic polyphosphate (polyP). AprA and polyP affected levels of more than 200 proteins, with an overlap of both upregulating 25 proteins and downregulating two proteins. Two proteins were upregulated by AprA but downregulated by polyP, while two others showed the opposite trend. Surprisingly, many of the AprA- and polyP-regulated proteins are associated with RNA metabolism and ribosomes. AprA increased phosphorylation of 15 proteins and decreased phosphorylation of 36 proteins. PolyP increased phosphorylation of 12 proteins and decreased phosphorylation of 12 proteins. As expected, the two chemorepellents affected phosphorylation of signal transduction/ motility proteins, but unexpectedly affected phosphorylation of RNA-associated proteins. Both AprA and polyP decreased phosphorylation of five proteins including the Ras-interacting protein RipA and guanine nucleotide exchange factors (GEFs) such as the RacGEF GxcT. Mutants lacking RipA or GxcT were unresponsive to both AprA and polyP chemorepulsion. Together, this work supports the idea that rather than activating the same chemorepulsion mechanism, AprA and polyP activate only partially overlapping chemorepulsion mechanisms, and identifies two new components that are used by both chemorepellents.

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 perform 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.

Modern Methods of Inorganic Polyphosphate Analysis in Biological Samples (Review)

The physiological phosphates (PPs) are crucial in various biochemical pathways and bioenergetics. Their dysregulation is associated with multiple cellular dysfunctions and life-threatening diseases. Designing sensitive and selective bioresponsive probes capable of differentiating Pi/NPPs levels is crucial for diagnostics and therapy. Developing probes for selective discrimination of phosphates faces intrinsic challenges due to their high hydration energy (ΔGhyd) barrier and structural similarity. The hard Ln(III) ions can overcome the high ΔGhyd barrier and are ideal for the detection and discrimination of PPs due to their longer lifetimes, using time-resolved luminescence (TRL) assays. Herein, two water-soluble luminescent Eu(III) probes, [Eu.L1] and [Eu.L2], can discriminate physiological inorganic (Pi) and nucleoside polyphosphates (NPPs) in H2O based on their differential binding interaction with the respective probe, triggering analyte-specific energy-transfer pathways. We established the involvement of coordinative unsaturation and π-π stacking interaction between the [Eu.L] probes and PPs, which modulates the nonradiative vibrational energy transfer (VET) and photo-induced electron transfer (PeT) pathways, responsible for their specific luminescence response and selectivity. The interaction of phosphates with the Eu(III) probes were monitored by TRL spectroscopy, lifetime (τ) and hydration state (q) measurements, and 31P NMR spectroscopy. These results connect the intricate molecular interactions of the [Eu.L] probes with PPs with concomitant TRL-responses.

Tuberculosis (TB) is one of the oldest infectious diseases known to humankind, with traces of its presence found in remains that are around 17,000 years old. TB is mostly caused by the tiny aerobic non-motile bacillus Mycobacterium TB (MTB). The unique shape and chemical content of the mycobacterial cell wall make an efficient TB therapy method challenging. A strict bacterial survival strategy for establishing drug tolerance in the stringent response (SR), MTB is a sophisticated remodeling of metabolism that slows down growth and energy requirements during famine. Recent studies emphasize the need to focus on the SR in MTB as a means of reducing the treatment duration. The MTB genome codes two polyphosphate kinases (PPK-1 and PPK-2), for maintenance of intracellular Inorganic Polyphosphate (Poly P) levels. The identification of a virulence factor of TB growth as well as persistence in host tissues may be helped in MTB using PPK2, which is required to modulate intracellular levels of regulating molecules and to sustain sensitivity to the first-line anti-drug isoniazid. Synthesized and under control by PPK2 enzymes, inorganic polyP is essential in this process since it controls stress reactions. This research, therefore, investigates the significance of PPK2 in the MTB, the chemicals suppressing a bacterial SR in MTB, and the list of PPK2 inhibitors for shortening TB.

Chemorepulsion causes cells to move away from the source of a signal, but the underlying mechanisms for eukaryotic cells are poorly understood. We performed proteomics and phosphoproteomics to elucidate how Dictyostelium discoideum responds to its two endogenous chemorepellent signals, the protein AprA and inorganic polyphosphate (polyP). At 60 minutes, AprA increased levels of 211 proteins and reduced levels of 57 proteins while polyP increased levels of 152 proteins and reduced levels of 168 proteins. Surprisingly, many of the AprA- and polyP-regulated proteins are associated with RNA metabolism and ribosomes. AprA and polyP both upregulated 19 proteins, one protein was downregulated by both, and one was upregulated by AprA and downregulated by polyP. AprA increased phosphorylation of 12 proteins and decreased phosphorylation of 60 proteins. PolyP increased phosphorylation of 7 proteins and decreased phosphorylation of 18 proteins. As expected, the two chemorepellents affected phosphorylation of signal transduction/ motility proteins, but unexpectedly affected phosphorylation of RNA-associated proteins. Both AprA and polyP decreased phosphorylation of six proteins including the Ras-interacting protein RipA and guanine nucleotide exchange factors (GEFs) such as the RacGEF GxcT. Mutants lacking RipA or GxcT were unresponsive to both AprA and polyP chemorepulsion. Together, this work supports the idea that rather than activating the same chemorepulsion mechanism, AprA and polyP activate only partially overlapping chemorepulsion mechanisms, and identifies two new components that are used by both chemorepellents.

Myofibroblasts drive wound healing and fibrotic disease through generation of contractile force to promote wound closure and production of matrix proteins to generate scar tissue. Platelets secrete many pro-wound healing molecules, including cytokines and growth factors. We previously reported that inorganic polyphosphate, secreted by activated platelets, is chemotactic for fibroblasts and induces a myofibroblast phenotype. Using NIH-3T3 cells and primary human fibroblasts, we examined the impact of inhibitors of cell-surface receptors and intracellular signaling molecules on polyphosphate-induced myofibroblast differentiation. We now report that polyphosphate-induced differentiation of fibroblasts to myofibroblasts occurs through a signaling pathway mediated by the receptor for advanced glycation end products (RAGE) and nuclear factor kappa B (NFκB) transcription factor. Inhibition of these signaling components ablated the effects of polyphosphate on fibroblasts. Platelet releasates also induced NFκB signaling and myofibroblast differentiation. Blocking the polyphosphate content of platelet releasates with a biocompatible polyP inhibitor rendered the releasates unable to induce myofibroblast differentiation. These results identify a cell-surface receptor and intracellular transcription factor utilized by platelet polyphosphate to promote wound healing through myofibroblast differentiation and may provide targets for promoting wound healing or altering the disease progression of fibrosis.

β-Tricalcium phosphate (β-TCP) is widely used as a material for bone implants due to its excellent biocompatibility, biodegradability, and osteoconductivity, as well as its osteoinductive properties. Here, we demonstrate that the regenerative potential of this material can be significantly enhanced when incorporated into a matrix of inorganic polyphosphate (polyP), a physiological, metabolically active polymer composed of phosphate residues linked by high-energy phosphoanhydride bonds. A 3D-printable hydrogel was developed containing suspendedβ-TCP and amorphous calcium-polyP nanoparticles (Ca-polyP-NP; the water-insoluble depot form of polyP), as well as NaH2PO4as the monomeric precursor of the polymeric, water-soluble Na-polyP. Heating the printed scaffold to 700 °C causes condensation of NaH2PO4, resulting in the formation of a Na-polyP glass melt that embeds the Ca-polyP-NP andβ-TCP particles. The final scaffolds exhibited the necessary porosity, with pore sizes ranging from 10 to 100 µm (average 84 µm), which are suitable for bone ingrowth, along with the required mechanical stability. The morphogenetically active polyP component is released from the 3D-printed porous scaffolds in appropriate amounts, significantly increasing both the proliferation and energy-dependent differentiation of mesenchymal stem cells (MSCs) into mineralizing osteoblasts compared to polyP-freeβ-TCP scaffolds. Moreover, enhanced formation of collagen fibers and hydroxyapatite deposits on the cell surface, as well as accelerated microvessel tube formation, were observed in MSCs seeded on polyP-containing scaffolds. These results d`emonstrate that the novel strategy of integratingβ-TCP with polyP as an energy-supplying, regeneration-promoting component imparts superior functional properties toβ-TCP scaffolds, making them a promising material for future bone implant applications.

The molecular mechanism of P2Y1 receptor activation by inorganic polyphosphates.

Smart coacervate microdroplets: biomimetic design, material innovations, and emerging applications in biomacromolecule delivery.

Vaspin is highly expressed not only in the skin but also in the liver and adipose tissue. It counteracts inflammation and oxidative stress in inflammatory skin diseases, obesity, and associated metabolic disorders, in part by inhibiting the kallikrein proteases KLK7 and KLK14. Vaspin binds the cell-surface low-density lipoprotein receptor-related protein 1 (LRP1) with nanomolar affinity, and is rapidly internalized into adipocytes and other cells. We found intracellular vaspin partially localized in the nucleus. Since vaspin binds heparin and inorganic polyphosphates, we investigated the DNA binding of vaspin. Using DNA-affinity chromatography and differential radial capillary action of ligand assays, we found high-affinity binding to random sequences of single- and double-stranded DNA for both vaspin and KLK7. Furthermore, KLK7 inhibition was accelerated fivefold in the presence of DNA molecules at least 40 bases in length. We previously identified the heparin-binding site at a basic patch on the central beta-sheet A of vaspin. In the current work, we determined the crystal structure of polyphosphate P45-bound vaspin, which confirmed previously identified residues mutated to generate a nonheparin-binding (NHB) vaspin variant. While NHB vaspin failed to bind heparin and polyP45, it still bound DNA with high affinity and accelerated protease inhibition. Mutation of closely spaced basic residues in helix A and helix G did not significantly alter DNA binding. In conclusion, we have identified vaspin as the second human DNA-binding serpin. While the exact mode of the nonspecific interaction remains unclear, it accelerates protease inhibition and likely contributes to the nuclear localization observed for internalized vaspin and may allow for intracellular effects.

Chromosome transmission fidelity is vital for organism fitness. Yet, extrinsic and intrinsic changes can affect this process, leading to aneuploidy, the loss/gain of chromosomes, which is a hallmark of cancer. Here, using a haploid fission yeast Schizosaccharomyces pombe strain with a segmental aneuploidy, we assayed genome stability under different temperatures and altered gene dosage. We find that S. pombe genome stability is temperature-dependent and is unexpectedly modulated by intracellular levels of inorganic polyphosphate polymers (polyP). The vtc4+ gene, encoding a subunit of the polyP-generating VTC complex, is present twice due to the segmental aneuploidy resulting in a gene-dosage-coupled increase in polyP. Using strains with different amounts of polyP, we find a direct negative correlation between polyP and chromosome segregation fidelity. PolyP modulates the function of the conserved CCAN kinetochore subcomplex, as the abnormal growth phenotype caused by the mutant CCAN protein Fta2-291 was rescued in the absence of polyP, while extra polyP had the opposite effect. Importantly, this appears to occur in part by modulation of the nucleolin Gar2. Gar2 is the functional homolog of the Saccharomyces cerevisiae Nsr1 protein, whose function is modulated by posttranslational polyP-mediated polyphosphorylation. Thus, polyP modulates genome stability, linking cellular metabolism to chromosome transmission fidelity.

The ability of bacteria to absorb metal ions and phosphates allows the development of processes for bioremediation of the environment and wastewater from heavy metals and excess phosphates. In this work, the ability of the "iron bacterium" Sphaerotilus montanus VKM B-2519 to remove manganese, iron, and phosphate from culture medium was studied. The bacterium removed Mn2+ but not Fe2+ from the culture medium during growth. At an initial concentration of 3mmol/L Mn2+, about 40% of phosphate and no more than 10% of manganese remained in the medium after cultivation of S. montanus for the stationary stage. Inorganic polyphosphate did not contribute to the removal of phosphate by S. montanus. In the presence of excess Mn2+, S. montanus formed Mn3(PO4)2 as a precipitate. It was confirmed by both chemical analysis and EDX. In the presence of excess Mn2+, S. montanus secreted phosphorylated exopolysaccharide into the culture medium. The data suggested that S. montanus VKM B-2519 is a prospective species for developing phosphate and manganese removal and biological sedimentation of manganese phosphate.

Inorganic polyphosphate (iPolyP) is a ubiquitous molecule composed of a variable number of orthophosphate units. Recent studies have highlighted its involvement in colorectal cancer (CRC) cell proliferation. However, further investigations are needed to elucidate its role in CRC cell progression and migration, as well as its influence on the tumor microenvironment. This study focuses on the inorganic polyphosphate (iPolyP)/transient receptor potential cation channel subfamily M member 8 (TRPM8) axis and its impact on CRC progression. To investigate these issues, western blotting, fixed and live cells immunofluorescence, 2D and 3D cell culture on CRC-patient derived tissues, ELISA, and wound healing assays were performed. Our results show that inorganic polyphosphate induces the expression of epithelial-to-mesenchymal transition (EMT) markers in CRC cells. Furthermore, the iPolyP/TRPM8 axis indirectly promotes tumor growth through activation of the Nucleotide-binding oligomerization domain, Leucine-rich Repeat and Pyrin domain-containing protein 3 (NLRP3) inflammasome in immune cells, leading to increased levels of the pro-inflammatory cytokine interleukin-1β (IL-1β) in the tumor microenvironment (TME), thereby advancing CRC. These findings suggest that targeting the iPolyP/TRPM8 pathway may be a promising strategy to inhibit CRC progression and metastasis.

Ulcerative colitis (UC) poses a significant therapeutic challenge due to its complex pathogenesis and limited treatment efficacy. Restoring gut microbiota homeostasis is a critical strategy, given its profound influence on immunity and metabolic dysregulation in UC. This study demonstrates the therapeutic potential of inorganic polyphosphate (polyP) in ameliorating UC by modulating gut microbiota and metabolites. In vivo experiments demonstrate that polyP administration alleviates colitis pathology, reduces proinflammatory cytokines (IL-1β, TNF-α), and reinforces intestinal barrier integrity. PolyP restructures microbial communities by enriching probiotics (Ruminococcaceae, Butyricicoccus, Harryflintia) while depleting Parasutterella. Concurrently, it reprograms metabolomic profiles, elevating barrier-repairing metabolites─notably indoleacetaldehyde (IAAld). These findings establish polyP as a microbiota-modulating therapeutic agent for UC, highlighting its role in modeling host-microbiota-metabolite interactions for gut homeostasis.

Chitosan/ZIF-8 interface-modified ammonium polyphosphate as core-shell additive for synergistically enhancing flame retardancy and degradation performance of polylactic acid.

Factor XIIa (FXIIa) is generated from its zymogen factor XII (FXII) by contact with polyanions such as inorganic polyphosphates. FXIIa cleaves the substrates prekallikrein and factor XI, triggering inflammatory cascades and plasma coagulation. From the N-terminus, FXII has fibronectin type II (FnII), epidermal growth factor-1 (EGF1), fibronectin type I (FnI), EGF2 and kringle domains. The N-terminal domains of FXII mediate polyanion and Zn2+ binding. To understand how ligand binding to polyanions and Zn2+ is coordinated across multiple domains, we determined the crystal structure of recombinant FXII domains 1-5 (FXIIHC5) to 3.4 Å resolution. A separate crystal structure of the isolated FXII FnII domain at 1.2 Å resolution revealed two bound Zn2+ ions. In FXIIHC5 a head-to-tail interaction is formed between the FnII and kringle domains, co-localizing the lysine-binding sites of the kringle domain and the cation-binding site of the FnII domain. Two FXIIHC5 monomers interlock, burying a large surface area of 2067 Å2, such that two kringle domains point outwards separated by a distance of 20 Å. The polyanion-binding site in the EGF1 domain is localized onto a plane together with the FnII and FnI domains. Using native mass spectrometry, we detected a major FXIIHC5 monomer peak and a minor dimer peak. Small-angle X-ray scattering and gel-filtration chromatography revealed the presence of monomers and dimers in solution. These FXII N-terminal domain structures provide a holistic framework to understand how the mosaic domain structure of FXII assembles diverse ligand-binding sites in three dimensions.