High Concentrations Of ATP Research Articles

Vascular smooth muscle cell-derived exosomes promote osteoblast-to-osteocyte transition via β-catenin signaling

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via β-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/β-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.

Increased concentrations of P2X7R in oligodendrocyte derived extracellular vesicles of Multiple sclerosis patients

Activation of the purinergic receptor P2X7 (P2X7R) is believed to be deleterious in autoimmune diseases and it was hypothesized to play a role in the pathogenesis of MS. P2X7R is an ATP-gated non-selective cationic channel; its activation can be driven by high concentrations of ATP and leads to the generation of large, cytolytic conductance pores. P2X7R activation can also result in apoptosis as a consequence of the activation of the caspase cascade via P2X7R-dependent stimulation of the NLRP3 inflammasome. We measured P2X7R in oligodendrocyte derived extracellular vesicles (ODEVs) in MS patients and in healthy subjects.Sixty-eight MS patients (50 relapsing-remitting, RR-MS, 18 primary progressive, PP-MS) and 57 healthy controls (HC) were enrolled. ODEVs were enriched from serum by a double step immunoaffinity method using an anti OMGp (oligodendrocyte myelin glycoprotein) antibody. P2X7R concentration was measured in ODEVs lysates by ELISA.One–way Anova test showed that P2X7R in ODEVs is significantly higher in PP-MS (mean: 1742.89 pg/mL) compared both to RR-MS (mean: 1277.33 pg/mL) (p < 0.001) and HC (mean: 879.79 pg/mL) (p < 0.001). Comparison between RR-MS and HC was also statistically significant (p < 0.001). Pearson's correlations showed that P2RX7 in ODEVs was positively correlated with EDSS (p = 0.002, r = 0.38, 0.15–0.57 95% CI) and MSSS (p = 0.004, r = 0.34, 0.12–0.54 95% CI) scores, considering MS patients together (PP-MS + RR-MS) and with disease duration in PP-MS group (p = 0.02, r = 0.53, 0.09–0.80 95% CI).Results suggest that ODEVs-associated P2X7R levels could be a biomarker for MS.

Importance of Intracellular Energy Status on High-Hydrostatic-Pressure Inactivation of sake Yeast Saccharomyces cerevisiae.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

Elucidation of the mechanism of elicitation of edible sprouts using UV-C radiation

The major aim of this study was to demonstrate the influence of UV-C radiation exposure of the edible sprouts on their redox status and the mechanism of production of selected phytochemicals in them. The study has shown that UV-C irradiation of broccoli sprouts and red cabbage for 5 min, and alfalfa for 15 min activates mechanisms involved in the antioxidant protection of the cell, resulting in an increase in the level of low-molecular antioxidants, i.e. polyphenols and glutathione. It was proven that sprouts irradiated with UV-C were characterised by higher MAPK expression, as well as higher level of ABA (ROS-dependent) which contributed to the induction of transcription factors (MYB) and consequently, to increased expression of proteins involved in antioxidants biosynthesis (PAL, CHS, γ-GCS, GS) and antioxidant enzymes (MnSOD, CAT). Moreover, UV-C increased mitochondrial activity, which was observed by higher concentration of ATP, as well as increased expression of SnRK1 protein.

Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry.

Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes.

Expression of ADK-S and ADK-L Isoforms and Their Association with CD39/CD73/A2aR in Colorectal Cancer.

The level of mRNA of the long (L) and short (S) isoforms of adenosine kinase (ADK) was analyzed in patients with colorectal cancer (CRC). ADK is required to convert adenosine (ADO) to AMP. It was shown that tumor and normal colon tissues (n=13) do not differ in the level of ADK-S and ADK-L mRNA. At the same time, the level of ADK-S mRNA (tumor: p=0.0214, normal: p=0.005) in the colon tissue was lower than in the blood of CRC patients (n=20), and the level of ADK-L mRNA (tumor: p=0.007, normal: p=0.024), on the contrary, was higher. A negative correlation was found between the level of ADK-S mRNA and the level of A2aR mRNA in both tumor and normal tissues (p=0.018 and p=0.0014, respectively). In the tumor tissue, a positive correlation was shown between ADK-L and CD73 mRNA levels (p=0.017). The obtained data indicate the association ADK with the expression of CD39/CD73/A2aR in CRC patients. In this regard, the effect of recombinant ADK on the expression of CD39 and CD73 ectonucleotidase by T cells in vitro was analyzed. In a culture of peripheral blood mononuclear cells isolated from the blood of 5 healthy donors, ADK did not abolish the inhibitory effect on the expression of CD39 and CD73 by CD8+T cells in the presence of a high concentration of ATP (a source for ADO). Effects on CD39+CD4+, CD73+CD4+T cells and CD39+ Treg cells were also not found.

Assembly-mediated activation of the SIR2-HerA supramolecular complex for anti-phage defense

SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.

Interaction of calcium responsive proteins and transcriptional factors with the PHO regulon in yeasts and fungi.

Phosphate and calcium ions are nutrients that play key roles in growth, differentiation and the production of bioactive secondary metabolites in filamentous fungi. Phosphate concentration regulates the biosynthesis of hundreds of fungal metabolites. The central mechanisms of phosphate transport and regulation, mediated by the master Pho4 transcriptional factor are known, but many aspects of the control of gene expression need further research. High ATP concentration in the cells leads to inositol pyrophosphate molecules formation, such as IP3 and IP7, that act as phosphorylation status reporters. Calcium ions are intracellular messengers in eukaryotic organisms and calcium homeostasis follows elaborated patterns in response to different nutritional and environmental factors, including cross-talking with phosphate concentrations. A large part of the intracellular calcium is stored in vacuoles and other organelles forming complexes with polyphosphate. The free cytosolic calcium concentration is maintained by transport from the external medium or by release from the store organelles through calcium permeable transient receptor potential (TRP) ion channels. Calcium ions, particularly the free cytosolic calcium levels, control the biosynthesis of fungal metabolites by two mechanisms, 1) direct interaction of calcium-bound calmodulin with antibiotic synthesizing enzymes, and 2) by the calmodulin-calcineurin signaling cascade. Control of very different secondary metabolites, including pathogenicity determinants, are mediated by calcium through the Crz1 factor. Several interactions between calcium homeostasis and phosphate have been demonstrated in the last decade: 1) The inositol pyrophosphate IP3 triggers the release of calcium ions from internal stores into the cytosol, 2) Expression of the high affinity phosphate transporter Pho89, a Na+/phosphate symporter, is controlled by Crz1. Also, mutants defective in the calcium permeable TRPCa7-like of Saccharomyces cerevisiae shown impaired expression of Pho89. This information suggests that CrzA and Pho89 play key roles in the interaction of phosphate and calcium regulatory pathways, 3) Finally, acidocalcisomes organelles have been found in mycorrhiza and in some melanin producing fungi that show similar characteristics as protozoa calcisomes. In these organelles there is a close interaction between orthophosphate, pyrophosphate and polyphosphate and calcium ions that are absorbed in the polyanionic polyphosphate matrix. These advances open new perspectives for the control of fungal metabolism.

The P2X7 Receptor, a Multifaceted Receptor in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by impaired episodic memory and two pathological lesions: amyloid plaques and neurofibrillary tangles. In AD, damaged neurons and the accumulation of amyloid β (Aβ) peptides cause a significant release of high amounts of extracellular ATP, which acts as a danger signal. The purinergic receptor P2X7 is the main sensor of high concentrations of ATP, and P2X7 has been shown to be upregulated in the brains of AD patients, contributing to the disease's pathological processes. Further, there are many polymorphisms of the P2X7 gene that impact the risk of developing AD. P2X7 can directly modulate Aβ plaques and Tau protein lesions as well as the inflammatory response by regulating NLRP3 inflammasome and the expression of several chemokines. The significant role of microglial P2X7 in AD has been well established, although other cell types may also be important in P2X7-mediated mechanisms. In this review, we will discuss the different P2X7-dependent pathways involved in the development of AD.

The Pannexin 1 Channel and the P2X7 Receptor Are in Complex Interplay to Regulate the Release of Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

The bladder urothelium releases ATP into the lamina propria (LP) during filling, which can activate P2X receptors on afferent neurons and trigger the micturition reflex. Effective ATP concentrations are largely dependent on metabolism by membrane-bound and soluble ectonucleotidases (s-ENTDs), and the latter are released in the LP in a mechanosensitive manner. Pannexin 1 (PANX1) channel and P2X7 receptor (P2X7R) participate in urothelial ATP release and are physically and functionally coupled, hence we investigated whether they modulate s-ENTDs release. Using ultrasensitive HPLC-FLD, we evaluated the degradation of 1,N6-etheno-ATP (eATP, substrate) to eADP, eAMP, and e-adenosine (e-ADO) in extraluminal solutions that were in contact with the LP of mouse detrusor-free bladders during filling prior to substrate addition, as an indirect measure of s-ENDTS release. Deletion of Panx1 increased the distention-induced, but not the spontaneous, release of s-ENTDs, whereas activation of P2X7R by BzATP or high concentration of ATP in WT bladders increased both. In Panx1-/- bladders or WT bladders treated with the PANX1 inhibitory peptide 10Panx, however, BzATP had no effect on s-ENTDS release, suggesting that P2X7R activity depends on PANX1 channel opening. We concluded, therefore, that P2X7R and PANX1 are in complex interaction to regulate s-ENTDs release and maintain suitable ATP concentrations in the LP. Thus, while stretch-activated PANX1 hinders s-ENTDS release possibly to preserve effective ATP concentration at the end of bladder filling, P2X7R activation, presumably in cystitis, would facilitate s-ENTDs-mediated ATP degradation to counteract excessive bladder excitability.

Human soluble CD39 displays substrate inhibition in a substrate-specific manner

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD1) metabolizes extracellular ATP and ADP to AMP. AMP is subsequently metabolized by CD79 to adenosine. CD39 activity is therefore a key regulator of purinergic signalling in cancer, thrombosis, and autoimmune diseases. In this study we demonstrate that soluble, recombinant CD39 shows substrate inhibition with ADP or ATP as the substrate. Although CD39 activity initially increased with increasing substrate concentration, at high concentrations of ATP or ADP, CD39 activity was markedly reduced. Although the reaction product, AMP, inhibits CD39 activity, insufficient AMP was generated under our conditions to account for the substrate inhibition seen. In contrast, inhibition was not seen with UDP or UTP as substrates. 2-methylthio-ADP also showed no substrate inhibition, indicating the nucleotide base is an important determinant of substrate inhibition. Molecular dynamics simulations revealed that ADP can undergo conformational rearrangements within the CD39 active site that were not seen with UDP or 2-methylthio-ADP. Appreciating the existence of substrate inhibition of CD39 will help the interpretation of studies of CD39 activity, including investigations into drugs that modulate CD39 activity.

Smart Telomerase-Gated DNA cage for precise siRNA release in cancer cells

Oligonucleotide nanoparticles (ONPs) have been utilized as a small interfering RNA (siRNA) delivery system because of their multiple advantages. However, the release of siRNA by ONPs inner cells mainly relies on acidic environments and high concentrations of ATP. These conditions do not allow the specific release of siRNA in tumor cells, which would prevent the side effects that result from the release of siRNA in normal cells. In this study, a prism oligonucleotide nanoparticle (pONP)-based delivery system was designed, which targeted with telomeric primers (TS) that can be extended by cancer-specific telomerase for specifically releasing siRNA inside telomerase-positive cancer cells. In vitro tests confirmed that this cage facilitated the release of siRNA specifically into telomerase-positive HeLa and MDA-MB-231 tumor cells. The release was so precise that almost no siRNA was released into telomerase-negative cells. In vivo tests also confirmed that this cage could release sibcl-2 specifically in tumor cells in an MDA-MB-231 orthotopic breast cancer mouse model. Moreover, the cage has a long half-life (e.g., 2.83 h in FBS) in the circulatory system and protects the cargo from nuclease degradation; thus, the released sibcl-2 effectively silences the translation of target cancer-related mRNA (bcl-2), similar to the sibcl-2 delivered by Lipo6000 in the telomerase-positive environment of cancer cells. pONP/TS/siRNA is readily prepared using a simple sequence design together with one-step self-assembly. This nanoparticle cage is a promising delivery method for precise siRNA release into targeted telomerase-positive cancer cells.

Tumor Microenvironment-Responsive Hydrogel for Direct Extracellular ATP Imaging-Guided Surgical Resection with Clear Boundaries.

Most solid tumors are clinically treated using surgical resection, and the presence of residual tumor tissues at the surgical margins often determines tumor survival and recurrence. Herein, a hydrogel (AHB Gel) is developed for fluorescence-guided surgical resection. AHB Gel is constructed by tethering a polyacrylamide hydrogel and ATP-responsive aptamers together. It exhibits strong fluorescence under high ATP concentrations corresponding to the TME (100-500μM) but shows little fluorescence at low ATP concentrations (10-100nM) such as those in normal tissues. AHB Gel can rapidly (within 3min) emit fluorescence after exposure to ATP, and the fluorescence signal only occurs at sites exposed to high ATP, resulting in a clear boundary between the ATP-high and ATP-low regions. In vivo, AHB Gel exhibits specific tumor-targeting capacity with no fluorescence response in normal tissue, providing clear tumor boundaries. In addition, AHB Gel has good storage stability, which is conducive to its future clinical application. In summary, AHB Gel is a novel tumor microenvironment-targeted DNA-hybrid hydrogel for ATP-based fluorescence imaging. It could enable the precise imaging of tumor tissues, showing promising application in fluorescence-guided surgeries in the future. This article is protected by copyright. All rights reserved.

Role of creatine shuttle in colorectal cancer cells.

The creatine shuttle translocates the energy generated by oxidative phosphorylation to the cytoplasm via mitochondrial creatine kinase (MTCK) and creatine kinase B (CKB) in the cytoplasm. It is not apparent how the creatine shuttle is related to cancer. Here, we analyzed the expression and function of CKB and MTCK in colorectal cancer (CRC) and investigated the role of the creatine shuttle in CRC. Compared with normal mucosa, 184 CRC tissues had higher levels of CKB and MTCK, and these levels were associated with histological grade, tumor invasion, and distant metastasis. CK inhibitor dinitrofluorobenzene (DNFB) on CRC cell lines HT29 and CT26 inhibited cell proliferation and stemness to less than 2/3 and 1/20 of their control levels, respectively. In this treatment, the production of reactive oxygen species increased, mitochondrial respiration decreased, and mitochondrial volume and membrane potential decreased. In a syngeneic BALB/c mouse model using CT26 cells pretreated with DNFB, peritoneal metastasis was suppressed to 70%. Phosphorylation of EGFR, AKT, and ERK1/2 was inhibited in DNFB-treated tumors. High ATP concentrations prevented EGFR phosphorylation in HT29 cells following DNFB treatment, CKB or MTCK knockdown, and cyclocreatine administration. Despite not being immunoprecipitated, CKB and EGFR were brought closer together by EGF stimulation. These findings imply that blocking the creatine shuttle decreases the energy supply, suppresses oxidative phosphorylation, and blocks ATP delivery to phosphorylation signals, preventing signal transduction. These findings highlight the critical role of the creatine shuttle in cancer cells and suggest a potential new cancer treatment target.

The Antiparallel Coiled-Coil Domain Allows Multiple Forward Step Sizes of Myosin X.

Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of ∼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.

P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer.

Bladder cancer is amongst the most common causes of cancer death worldwide. Muscle-invasive bladder cancer (MIBC) bears a particularly poor prognosis. Overexpression of purinergic P2X receptors (P2XRs) has been associated with worse outcome in several malignant tumors. Here, we investigated the role of P2XRs in bladder cancer cell proliferation in vitro and the prognostic value of P2XR expression in MIBC patients. Cell culture experiments with T24, RT4, and non-transformed TRT-HU-1 cells revealed a link between high ATP concentrations in the cell culture supernatants of bladder cell lines and a higher grade of malignancy. Furthermore, proliferation of highly malignant T24 bladder cancer cells depended on autocrine signaling through P2X receptors. P2X1R, P2X4R, and P2X7R expression was immunohistochemically analyzed in tumor specimens from 173 patients with MIBC. High P2X1R expression was associated with pathological parameters of disease progression and reduced survival time. High combined expression of P2X1R and P2X7R increased the risk of distant metastasis and was an independent negative predictor of overall and tumor-specific survival in multivariate analyses. Our results suggest that P2X1R/P2X7R expression scores are powerful negative prognostic markers in MIBC patients and that P2XR-mediated pathways are potential targets for novel therapeutic strategies in bladder cancer.

Amphibian pore-forming protein βγ-CAT drives extracellular nutrient scavenging under cell nutrient deficiency

Nutrient acquisition is essential for animal cells. βγ-CAT is a pore-forming protein (PFP) and trefoil factor complex assembled under tight regulation identified in toad Bombina maxima. Here, we reported that B.maxima cells secreted βγ-CAT under glucose, glutamine, and pyruvate deficiency to scavenge extracellular proteins for their nutrient supply and survival. AMPK signaling positively regulated the expression and secretion of βγ-CAT. The PFP complex selectively bound extracellular proteins and promoted proteins uptake through endolysosomal pathways. Elevated intracellular amino acids, enhanced ATP production, and eventually prolonged cell survival were observed in the presence of βγ-CAT and extracellular proteins. Liposome assays indicated that high concentration of ATP negatively regulated the opening of βγ-CAT channels. Collectively, these results uncovered that βγ-CAT is an essential element in cell nutrient scavenging under cell nutrient deficiency by driving vesicular uptake of extracellular proteins, providing a new paradigm for PFPs in cell nutrient acquisition and metabolic flexibility.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase-separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact, ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. Additionally, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids which are found seeding-inefficient due to their reduced β-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity.

The classical DNA aptamer for adenosine and ATP has been the most used small molecule binding aptamer for biosensing, imaging, and DNA nanotechnology. This sequence has recurred multiple times in previous aptamer selections, and all previous selections used a high concentration of ATP as the target. Herein, two separate selections were performed using adenosine and ATP as targets. By pushing the target concentrations down to the low micromolar range, two new aptamers with Kd as low as 230 nM were obtained, showing around 30-fold higher affinity compared to the classical aptamer. The classical aptamer sequence still dominated the library in the early rounds of the selections, but it was suppressed in the later rounds. The new aptamers bind to one target molecule instead of two. Mutation studies confirmed their secondary structures and specific binding. Using the deep sequencing data from the selections, long-standing questions such as the existence of one-site aptamers and mutation distribution in the classical aptamer were addressed. Comparisons were made with previously reported DNA aptamers for ATP. Finally, a strand-displacement biosensor was tested showing selectivity for adenosine and its nucleotides.

Recombinant Analogs of Sea Anemone Kunitz-Type Peptides Influence P2X7 Receptor Activity in Neuro-2a Cells

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.