Feng et al. (2020) developed a simple, nondestructive, and cost-effective method to quantify polyphosphate (poly-P) in poly-P-accumulating organism (PAO)-enriched sludge samples through 30-h ...anaerobic exposure to 1 % (w/v) ethylenediaminetetraacetic acid (EDTA). This study optimized the N/P ratio (∼2) of the PAO culture medium in order to provide excess P for poly-P formation in PAO cells. Subsequently, the fluorescence microscopic observation of stained cells confirmed that Corynebacterium glutamicum was a PAO species capable of heterotrophic nitrification. Finally, this study reevaluated the accuracy and specificity of the EDTA-based quantification method, using two confirmed PAO biomass, three confirmed non-PAO biomass, and two sludge samples. The 1 % (w/v) EDTA treatment appears destructive to non-PAO cells, causes the release of other P forms, and is not effective for all PAO species. Under the conditions, the actual P release amount should be calculated by subtracting approximately 8 mg P g−1 total suspended solids from the determination. The amounts of P released from sludge samples was determined not only by the PAO fractions described by Feng et al. but also by PAO community structure and sludge P content.
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•It led to underestimation (relative to PAO) or overestimation (relative to non-PAO) of P release.•It can be conducted using simpler classic anaerobic methods.•PAO community structure and sludge P content also determine the P release amount from sludge.
Summary
Factor Va enhances the rate of prothrombin activation by factor Xa by four to five orders of magnitude. Production of initiating levels of factor Va from its precursor, factor V, is a ...critical event early in haemostasis, as factor V exhibits negligible cofactor activity. While thrombin is the most potent physiological back-activator of factor V, the first prothrombinase complexes require a source of factor Va prior to thrombin generation. A recent study by Whelihan et al. (J Thromb Haemost 2010; 8:1532–1539) identified factor XIa as a candidate for the initial thrombin-independent activation of factor V, although this reaction was slow and required relatively high concentrations of factors V and XIa. Activated platelets secrete polyphosphate, which we previously showed to be potently procoagulant. We now report that polyphosphate greatly accelerates factor V activation by factor XIa, and that this is supported by polyphosphate polymers of the size secreted by activated human platelets. This finding provides additional evidence that factor XIa-mediated generation of factor Va may contribute to the initiation of haemostasis.
The initial mineralization centers during human bone formation onto osteoblasts are composed of CaCO3. Those bioseeds are enzymatically formed via carbonic anhydrase(s) in close association with the ...cell surface of the osteoblasts. Subsequently, the bicarbonate/carbonate anions are exchanged non‐enzymatically by inorganic phosphate Pi. One source for the supply of Pi is polyphosphate polyP which is a physiological polymer, formed in the osteoblasts as well as in the platelets. The energy‐rich acid anhydride bonds within the polyP chain are cleaved by phosphatase(s); during this reaction free‐energy might be released that could be re‐used, as metabolic fuel, for the maintenance of the steady‐state concentrations of the substrates/products during mineralization. Finally it is outlined that polyP, as a morphogenetically active scaffold, is even suitable for 3D cell printing.
Polyphosphate (polyP) is a natural inorganic polymer that is synthesized in cells in close vicinity of bone tissue. This polymer is biocompatible and bioresorbable. The characteristic features of polyP are the physiological origin and the huge conserved reservoir of metabolic energy. Since Ca‐polyP is built of phosphate and Ca2+, it can be fabricated to bone scaffolds.
Ionically crosslinked nanoparticles based on high and low molecular weight chitosans (CS) were formulated with plasmid DNA or dsDNA oligomers using the ionic gelation technique with pentasodium ...tripolyphospate (TPP) as crosslinking agent. The resulting CS/TPP nanoparticles were investigated with regard to their physical–chemical properties,
in vitro transfection efficiency, toxicity, cellular uptake, and
in vivo gene expression following intratracheal administration to mice. The effects of co-formulating the nanoparticles with a model protein, BSA, were also studied. CS/TPP nanoparticles showed high encapsulation efficiencies both for plasmid DNA and dsDNA oligomers (20-mers), independent of CS molecular weight. TEM images revealed a spherical shape of the CS/TPP nanoparticles in contrast to the heterogeneous and irregular morphology displayed by conventional chitosan polyplexes. The nanoparticles showed high physical stability and no DNA release could be detected in diverse release media, nor even after incubation with heparin. Low molecular weight (LMW) CS/TPP nanoparticles gave high gene expression levels in HEK 293 cells already 2 days after transfection, reaching a plateau of sustained and high gene expression between 4 and 10 days. The inclusion of BSA into the nanostructures did not alter the inherent transfection efficiency of the nanoparticles. Confocal studies suggest endocytotic cellular uptake of the nanoparticles and a subsequent release into the cytoplasm within 14
h. LMW CS/TPP nanoparticles mediated a strong beta-galactosidase expression
in vivo after intratracheal administration. The results of this study forward ionically crosslinked CS/TPP nanoparticles as a biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.
Nucleoside triphosphates play a central role in biology, but efforts to study these roles have proven difficult because the levels of triphosphates are tightly regulated in a cell and because ...individual triphosphates can be difficult to label or modify. In addition, many synthetic biology efforts are focused on the development of unnatural nucleoside triphosphates that perform specific functions in the cellular environment. In general, both of these efforts would be facilitated by a general means to directly introduce desired triphosphates into cells. Previously, we demonstrated that recombinant expression of a nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2) in Escherichia coli functions to import triphosphates that are added to the media. Here, to explore the generality and utility of this approach, we report a structure–activity relationship study of PtNTT2. Using a conventional competitive uptake inhibition assay, we characterize the effects of nucleobase, sugar, and triphosphate modification, and then develop an LC-MS/MS assay to directly measure the effects of the modifications on import. Lastly, we use the transporter to import radiolabeled or 2′-fluoro-modified triphosphates and quantify their incorporation into DNA and RNA. The results demonstrate the general utility of the PtNTT2-mediated import of natural or modified nucleoside triphosphates for different molecular or synthetic biology applications.
Bacteria are prime cell factories that can efficiently convert carbon and nitrogen sources into a large diversity of intracellular and extracellular biopolymers, such as polysaccharides, polyamides, ...polyesters, polyphosphates, extracellular DNA and proteinaceous components. Bacterial polymers have important roles in pathogenicity, and their varied chemical and material properties make them suitable for medical and industrial applications. The same biopolymers when produced by pathogenic bacteria function as major virulence factors, whereas when they are produced by non-pathogenic bacteria, they become food ingredients or biomaterials. Interdisciplinary research has shed light on the molecular mechanisms of bacterial polymer synthesis, identified new targets for antibacterial drugs and informed synthetic biology approaches to design and manufacture innovative materials. This Review summarizes the role of bacterial polymers in pathogenesis, their synthesis and their material properties as well as approaches to design cell factories for production of tailor-made bio-based materials suitable for high-value applications.
The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate‐limitations can be at the mono‐, but also at the di‐ and triphosphorylation steps. We ...developed a nucleoside triphosphate (NTP) delivery system (TriPPPro‐approach). In this approach, NTPs are masked by two bioreversible units at the γ‐phosphate. Using a procedure involving H‐phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme‐triggered delivery of NTPs was demonstrated by pig liver esterase, in human T‐lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro‐compounds of some HIV‐inactive nucleoside analogues showed marked anti‐HIV activity. For cellular uptake studies, a fluorescent TriPPPro‐compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.
NTP drugs go pro: The TriPPPro‐approach was used to synthesize a series of nucleoside triphosphate analogues. The TriPPPro‐compounds displayed anti‐HIV activity and cellular uptake. In some cases, even inactive parent nucleosides were converted into powerful antiviral compounds. These results could thus aid the development of future nucleoside prodrugs.
Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. ...described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue.
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•Lysine polyphosphorylation of Top1 and Nsr1 is a non-covalent modification•Lysine polyphosphorylation is ionic and sensitive to salt and protonation state•Lysine residues alone are sufficient for non-covalent polyP modification•Non-PASK yeast proteins undergo lysine-mediated non-covalent polyP modification
Lysine polyphosphorylation was posited to be a covalent linkage of polyphosphate (polyP) to lysine sidechains within proteins. Neville et al. find that this modification is non-covalent and sensitive to ionic strength and alkaline pH. Lysine residues alone drive polyP modification via electrostatic interaction with polyP.
Summary
Phosphate (Pi) availability is a major factor limiting plant growth and development. The key transcription factor controlling Pi‐starvation response (PSR) is PHOSPHATE STARVATION RESPONSE 1 ...(PHR1) whose transcript levels do not change with changes in Pi levels. However, how PHR1 stability is regulated at the post‐translational level is relatively unexplored in Arabidopsis thaliana.
Inositol polyphosphates (InsPn) are important signal molecules that promote the association of stand‐alone SPX domain proteins with PHR1 to regulate PSR. Here, we show that NITROGEN LIMITATION ADAPTATION (NLA) E3 ligase can associate with PHR1 through its conserved SPX domain and polyubiquitinate PHR1 in vitro. The association with PHR1 and its ubiquitination is enhanced by InsP6 but not by InsP5.
Analysis of InsPn‐related mutants and an overexpression plant shows PHR1 levels are more stable in itpk4‐1 and vih2‐4/VIH1amiRNA but less stable in ITPK4 overexpression plants. Under Pi‐deficient conditions, nla seedlings contain high PHR1 levels, display long root hair and accumulate anthocyanin in shoots phenocopying PHR1 overexpression plants. By contrast, NLA overexpression plants phenocopy phr1 whose phenotypes are opposite to those of nla.
Our results suggest NLA functions as a negative regulator of Pi response by modulating PHR1 stability and the NLA/PHR1 association depends on InsPn levels.
Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known ...as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.
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