Characterization of an extremophile bacterial acid phosphatase derived from metagenomics analysis.

Acid phosphatases are enzymes that play a crucial role in the hydrolysis of various organophosphorous molecules. A putative acid phosphatase called FS6 was identified using genetic profiles and sequences from different environments. FS6 showed high sequence similarity to type C acid phosphatases and retained more than 30% of consensus residues in its protein sequence. A histidine-tagged recombinant FS6 produced in Escherichia coli exhibited extremophile properties, functioning effectively in a broad pH range between 3.5 and 8.5. The enzyme demonstrated optimal activity at temperatures between 25 and 50°C, with a melting temperature of 51.6°C. Kinetic parameters were determined using various substrates, and the reaction catalysed by FS6 with physiological substrates was at least 100-fold more efficient than with p-nitrophenyl phosphate. Furthermore, FS6 was found to be a decamer in solution, unlike the dimeric forms of crystallized proteins in its family.

Short-term responses of soil enzyme activities and stoichiometry to litter input in Castanopsis carlesii and Cunninghamia lanceolata plantations.

Litter input triggers the secretion of soil extracellular enzymes and facilitates the release of carbon (C), nitrogen (N), and phosphorus (P) from decomposing litter. However, how soil extracellular enzyme activities were controlled by litter input with various substrates is not fully understood. We examined the activities and stoichiometry of five enzymes including ß-1,4-glucosidase, ß-D-cellobiosidase, ß-1,4-N-acetyl-glucosaminidase, leucine aminopeptidase and acidic phosphatase (AP) with and without litter input in 10-year-old Castanopsis carlesii and Cunninghamia lanceolata plantations monthly during April to August, in October, and in December 2021 by using an in situ microcosm experiment. The results showed that: 1) There was no significant effect of short-term litter input on soil enzyme activity, stoichiometry, and vector properties in C. carlesii plantation. In contrast, short-term litter input significantly increased the AP activity by 1.7% in May and decreased the enzymatic C/N ratio by 3.8% in August, and decreased enzymatic C/P and N/P ratios by 11.7% and 10.3%, respectively, in October in C. lanceolata plantation. Meanwhile, litter input increased the soil enzymatic vector angle to 53.8° in October in C. lanceolata plantations, suggesting a significant P limitation for soil microorganisms. 2) Results from partial least squares regression analyses showed that soil dissolved organic matter and microbial biomass C and N were the primary factors in explaining the responses of soil enzymatic activity to short-term litter input in both plantations. Overall, input of low-quality (high C/N) litter stimulates the secretion of soil extracellular enzymes and accelerates litter decomposition. There is a P limitation for soil microorganisms in the study area.

Isolation and biochemical characterization of novel acid phosphatase and zinc-dependent acid phosphatase from the chicken's brain.

The AcPase exhibits a specific activity of 31.32 U/mg of protein with a 728-fold purification, and the yield of the enzyme is raised to 3.15 %. The Zn2+-dependent AcPase showed a purification factor of 1.34 specific activity of 14 U/mg of proteins and a total recovery of 5.14. The SDS-PAGE showed a single band corresponding to a molecular weight of 18 kDa of AcPase and 29 kDa of Zn2+-dependent AcPase. The AcPase enzyme has shown a wide range of substrate specificity for p-NPP, phenyl phosphate and FMN, while in the case of ZnAcPase α and ß-Naphthyl phosphate and p-NPP were proved to be superior substrates. The divalent metal ions like Mg2+, Mn2+, and Ca2+ increased the activity, while other substrates decreased the enzyme activity. The Km (0.14 mM) and Vmax (21 µmol/min/mg) values of AcPase were higher than those of Zn2+-AcPase (Km = 0.5 mM; Vmax = 9.7 µmol/min/mg). The Zn2+ ions activate the Zn2+-AcPase while Fe3+, Al3+, Pb2+, and Hg2+ showed inhibition on enzyme activity. Molybdate, vanadate and phosphate were found to be competitive inhibitors of AcPase with Ki values 316 µM, 185 µM, and 1.6 mM, while in Zn2+-AcPase tartrate and phosphate also showed competitive inhibition with Ki values 3 mM and 0.5 mM respectively.

An off-on fluorescence method for acid phosphatase assay based on the inner filter effect of MnO2 nanosheets on vitamin B2.

Fluorescence analysis has attracted much attention due to its rapidity and sensitivity. The present work describes a novel fluorescence detection method for acid phosphatase (ACP) on the basis of inner-filter effect (IFE), where MnO2 nanosheets (MnO2 NSs) and vitamin B2 (VB2) are served as absorbers and fluorophores, respectively. In the absence of ACP, the absorption band of MnO2 NSs overlaps well with the excitation band of VB2, resulting in effective IFE and inhibition of VB2 fluorescence. In the presence of ACP, 2-phospho-L-ascorbic acid trisodium salt (AAP) is hydrolyzed to generate ascorbic acid (AA), which efficiently trigger the reduction of MnO2 NSs into Mn2+ ions, causing the weakening of the MnO2 NSs absorption band and the recovery of VB2 fluorescence. Further investigation indicates that the fluorescence recovery degree of VB2 increases with the increase of ACP concentration. Under selected experimental conditions, the proposed method can achieve sensitive detection of ACP in the ranges of 0.5-4.0 mU/mL and 4.0-15 mU/mL along with a limit of detection (LOD) as low as 0.14 mU/mL. Finally, this method was successfully applied for the detection of ACP in human serum samples with satisfactory recoveries in the range of 95.0 %-108 %.

Acid phosphatase involved in phosphate homeostasis in Brassica napus and the functional analysis of BnaPAP10s.

Purple acid phosphatases (PAPs) are involved in activating the rhizosphere's organic phosphorus (P) and promoting P recycling during plant development, especially under the long-term P deficiency conditions in acid soil. However, the function of BnaPAPs in response to P deficiency stress in Brassica napus has rarely been explored. In this study, we found that the acid phosphatase activities (APA) of rapeseed shoot and root increased under P deficienct conditions. Genome-wide identification found that 82 PAP genes were unevenly distributed on 19 chromosomes in B. napus, which could be divided into eight subfamilies. The segmental duplication events were the main driving force for expansion during evolution, and the gene structures and conserved motifs of most members within the same subfamily were highly conservative. Moreover, the expression levels of 37 and 23 different expressed genes were induced by low P in leaf and root, respectively. BnaA09.PAP10a and BnaC09.PAP10a were identified as candidate genes via interaction networks. Significantly, both BnaPAP10a overexpression lines significantly increased root-related APA and total phosphate concentration under P deficiency and ATP supply conditions, thereby improving plant growth and root length. In summary, our results provided a valuable foundation for further study of BnaPAP functions.

Purification, identification and Cryo-EM structure of prostatic acid phosphatase in human semen.

Prostatic acid phosphatase (PAP) is a glycoprotein that plays a crucial role in the hydrolysis of phosphate ester present in prostatic exudates. It is a well-established indicator for prostate cancer due to its elevated serum levels in disease progression. Despite its abundance in semen, PAP's influence on male fertility has not been extensively studied. In our study, we report a significantly optimized method for purifying human endogenous PAP, achieving remarkably high efficiency and active protein recovery rate. This achievement allowed us to better analyze and understand the PAP protein. We determined the cryo-electron microscopic (Cryo-EM) structure of prostatic acid phosphatase in its physiological state for the first time. Our structural and gel filtration analysis confirmed the formation of a tight homodimer structure of human PAP. This functional homodimer displayed an elongated conformation in the cryo-EM structure compared to the previously reported crystal structure. Additionally, there was a notable 5-degree rotation in the angle between the α domain and α/ß domain of each monomer. Through structural analysis, we revealed three potential glycosylation sites: Asn94, Asn220, and Asn333. These sites contained varying numbers and forms of glycosyl units, suggesting sugar moieties influence PAP function. Furthermore, we found that the active sites of PAP, His44 and Asp290, are located between the two protein domains. Overall, our study not only provide an optimized approach for PAP purification, but also offer crucial insights into its structural characteristics. These findings lay the groundwork for further investigations into the physiological function and potential therapeutic applications of this important protein.

SlPHL1 positively modulates acid phosphatase in response to phosphate starvation by directly activating the genes SlPAP10b and SlPAP15 in tomato.

Increased acid phosphatase (APase) activity is a prominent feature of tomato (Solanum lycopersicum) responses to inorganic phosphate (Pi) restriction. SlPHL1, a phosphate starvation response (PHR) transcription factor, has been identified as a positive regulator of low Pi (LP)-induced APase activity in tomato. However, the molecular mechanism underlying this regulation remains to be elucidated. Here, SlPHL1 was found to positively regulate the LP-induced expression of five potential purple acid phosphatase (PAP) genes, namely SlPAP7, SlPAP10b, SlPAP12, SlPAP15, and SlPAP17b. Furthermore, we provide evidence that SlPHL1 can stimulate transcription of these five genes by binding directly to the PHR1 binding sequence (P1BS) located on their promoters. The P1BS mutation notably weakened SlPHL1 binding to the promoters of SlPAP7, SlPAP12, and SlPAP17b but almost completely abolished SlPHL1 binding to the promoters of SlPAP10b and SlPAP15. As a result, the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 was substantially diminished. In addition, not only did transient overexpression of either SlPAP10b or SlPAP15 in tobacco leaves increase APase activity, but overexpression of SlPAP15 in Arabidopsis and tomato also increased APase activity and promoted plant growth. Subsequently, two SPX proteins, SlSPX1 and SlSPX4, were shown to physically interact with SlPHL1. Moreover, SlSPX1 inhibited the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 and negatively regulated the activity of APase. Taken together, these results demonstrate that SlPHL1-mediated LP signaling promotes APase activity by activating the transcription of SlPAP10b and SlPAP15, which may provide valuable insights into the mechanisms of tomato response to Pi-limited stress.

Inhibition effect of epicatechin gallate on acid phosphatases from rainbow trout (Oncorhynchus mykiss) liver by multispectral and molecular docking.

Inhibition of acid phosphatase, which significantly contributes to inosine 5'-monophosphate (IMP) degradation, is crucial for preventing flavor deterioration of aquatic products during storage. In this study, the inhibitory effect of epicatechin gallate (ECG) on the activity of acid phosphatase isozymes (ACPI and ACPII) was analyzed using inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry, and molecular simulation. ACPI and ACPII with molecular weights of 59.5 and 37.3 kDa, respectively, were purified from rainbow trout liver. ECG reversibly inhibited ACPI and ACPII activities via mixed-type inhibition, with half maximal inhibitory concentration (IC50) of 0.24 ± 0.01 mmol/L and 0.27 ± 0.03 mmol/L, respectively. Fluorescence spectra indicated that ECG statically quenched the intrinsic fluorescence of ACPI and ACPII. ECG could spontaneously bind to ACPI and ACPII through hydrogen bonding and van der Waals forces and exhibited a higher affinity for ACPI than for ACPII. In addition, molecular dynamic simulation revealed that ECG-ACPI and ECG-ACPII complexes were relatively stable during the entire simulation process. Our findings provide a theoretical basis for the use of ECG as an inhibitor of ACP to improve the flavor of aquatic products.

Mycorrhizal fungus Serendipita indica-associated acid phosphatase rescues the phosphate nutrition with reduced arsenic uptake in the host plant under arsenic stress.

Symbiotic interactions play a vital role in maintaining the phosphate (Pi) nutrient status of host plants and providing resilience during biotic and abiotic stresses. Serendipita indica, a mycorrhiza-like fungus, supports plant growth by transporting Pi to the plant. Despite the competitive behaviour of arsenate (AsV) with Pi, the association with S. indica promotes plant growth under arsenic (As) stress by reducing As bioavailability through adsorption, accumulation, and precipitation within the fungus. However, the capacity of S. indica to enhance Pi accumulation and utilization under As stress remains unexplored. Axenic studies revealed that As supply significantly reduces intracellular ACPase activity in S. indica, while extracellular ACPase remains unaffected. Further investigations using Native PAGE and gene expression studies confirmed that intracellular ACPase (isoform2) is sensitive to As, whereas extracellular ACPase (isoform1) is As-insensitive. Biochemical analysis showed that ACPase (isoform1) has a Km of 0.5977 µM and Vmax of 0.1945 Unit/min. In hydroponically cultured tomato seedlings, simultaneous inoculation of S. indica with As on the 14thday after seed germination led to hyper-colonization, increased root/shoot length, biomass, and induction of ACPase expression and secretion under As stress. Arsenic-treated S. indica colonized groups (13.33 µM As+Si and 26.67 µM As+Si) exhibited 8.28-19.14 and 1.71-3.45-fold activation of ACPase in both rhizospheric media and root samples, respectively, thereby enhancing Pi availability in the surrounding medium under As stress. Moreover, S. indica (13.33 µM As+Si and 26.67 µM As+Si) significantly improved Pi accumulation in roots by 7.26 and 9.46 times and in shoots by 4.36 and 8.85 times compared to the control. Additionally, S. indica induced the expression of SiPT under As stress, further improving Pi mobilization. Notably, fungal colonization also restricted As mobilization from the hydroponic medium to the shoot, with a higher amount of As (191.01 ppm As in the 26.67 µM As+Si group) accumulating in the plant's roots. The study demonstrates the performance of S. indica under As stress in enhancing Pi mobilization while limiting As uptake in the host plant. These findings provide the first evidence of the As-Pi interaction in the AM-like fungus S. indica, indicating reduced As uptake and regulation of PHO genes (ACPase and SiPT genes) to increase Pi acquisition. These data also lay the foundation for the rational use of S. indica in agricultural practices.

Multi-imaging platform for rhizosphere studies: Phosphorus and oxygen fluxes.

Rhizosphere is a soil volume of high spatio-temporal heterogeneity and intensive plant-soil-microbial interactions, for which visualization and process quantification is of highest scientific and applied relevance, but still very challenging. A novel methodology for quick assessment of two-dimensional distribution of available phosphorus (P) in rhizosphere was suggested, tested, and development up to the application platform. Available P was firstly trapped by an in-situ diffusive gradients in thin-films (DGT) sampler with precipitated zirconia as the binding gel, and subsequently, the loaded gel was analyzed with an optimized colorimetric imaging densitometry (CID). The imaging platform was established linking: i) DGT, ii) planar optode, and iii) soil zymography techniques to simultaneously determine available P, oxygen, and acid phosphatase in rhizosphere at sub-millimeter spatial scales. The DGT identified available P level in rice rhizosphere were spatially overlapping to the localized redox hotspots and phosphatase activity. The spatial relationship between available P and acid phosphatase activity was dependent on root development. The root radial oxygen loss (ROL) remained active during the experimental observations (2-3 days), while a flux of available P of 10 pg cm-2 s-1 was visualized within 2-3 mm of roots, confirming the correlative response of rice roots to oxygen secretion and P uptake. Summarizing, the established imaging platform is suitable to capture spatial heterogeneity and temporal dynamics of root activities, nutrient bioavailability, ROL and enzyme activities in rhizosphere.

Multi-omics analysis reveals the roles of purple acid phosphatases in organic phosphorus utilization by the tropical legume Stylosanthes guianensis.

Stylo (Stylosanthes guianensis) is a tropical legume known for its exceptional tolerance to low phosphate (Pi), a trait believed to be linked to its high acid phosphatase (APase) activity. Previous studies have observed genotypic variations in APase activity in stylo; however, the gene encoding the crucial APase responsible for this variation remains unidentified. In this study, transcriptomic and proteomic analyses were employed to identify eight Pi starvation-inducible (PSI) APases belonging to the purple APase (PAP) family in the roots of stylo and seven in the leaves. Among these PSI-PAPs, SgPAP7 exhibited a significantly positive correlation in its expression levels with the activities of both internal APase and root-associated APase across 20 stylo genotypes under low-Pi conditions. Furthermore, the recombinant SgPAP7 displayed high catalytic activity toward adenosine 5'-diphosphate (ADP) and phosphoenolpyruvate (PEP) in vitro. Overexpression (OE) of SgPAP7 in Arabidopsis facilitated exogenous organic phosphorus utilization. Moreover, SgPAP7 OE lines showed lower shoot ADP and PEP levels than the wild type, implying that SgPAP7 is involved in the catabolism and recycling of endogenous ADP and PEP, which could be beneficial for plant growth in low-Pi soils. In conclusion, SgPAP7 is a key gene with a major role in stylo adaptation to low-Pi conditions by facilitating the utilization of both exogenous and endogenous organic phosphorus sources. It may also function as a PEP phosphatase involved in a glycolytic bypass pathway that minimizes the need for adenylates and Pi. Thus, SgPAP7 could be a promising target for improving tolerance of crops to low-Pi availability.

Investigating the toxic effects of ethoprophos on Eisenia fetida: Integrating light microscopy, scanning electron microscopy, and biochemical analysis.

This research investigated the ecological impact of exposing Eisenia fetida, an essential component of soil ecosystems, to the organophosphate pesticide ethoprophos, widely used in agriculture. With a focus on understanding the specific effects on earthworms, we employed three concentrations (7.5, 15, and 30 mg/kg) over 28 days, considering the pesticide's short half-life and existing data on environmental concentrations. We aimed to contribute to a broader understanding of how these pesticides affect soil health. Histological analysis, including staining with Hematoxylin-eosin, Mallory Trichrome, Periodic acid-Schiff, and Alcian blue methods, was conducted on control and treatment groups. The histological and histopathological results were evaluated using the light microscopy, revealing various degenerations in the epithelial and muscle layers. Scanning electron microscopy analysis detected concentration-related notable compaction of the body surface, asymmetry, and distortion in the body segments. In the exposed groups, especially those subjected to higher ethoprophos concentrations, the grid-like appearance of the clitellum was visibly disturbed. This disturbance in the grid-like pattern is indicative of structural changes and disruptions at the microscopic level. Furthermore, total protein, carbohydrate, lipid analyses, as well as acid phosphatase and alkaline phosphatase enzyme activities, were also evaluated for earthworms from each experimental group. The analyses showed a concentration-related decrease in all biochemical measurements, except acid phosphatase enzyme activity. In conclusion, our study reveals that the environmentally realistic concentrations of ethoprophos, an effective and widely used pesticide in pest control, have detrimental effects on the health and physiology of E. fetida. These effects are manifested through histological deformities, altered biochemical profiles, and observable physiological disturbances. These results shed light on the harmful effects of ethoprophos on earthworms, underlining the necessity to restrict its usage in agricultural practices and thereby support environmental sustainability.

Phosphorus deficiency alters root length, acid phosphatase activity, organic acids, and metabolites in root exudates of soybean cultivars.

Phosphorus (P) deficiency alters the root morphological and physiological traits of plants. This study investigates how soybean cultivars with varying low-P tolerance values respond to different P levels in hydroponic culture by assessing alterations in root length, acid phosphatase activity, organic acid exudation, and metabolites in root exudates. Three low-P-tolerant cultivars ('Maetsue,' 'Kurotome,' and 'Fukuyutaka') and three low-P-sensitive cultivars ('Ihhon,' 'Chizuka,' and 'Komuta') were grown under 0 (P0) and 258 µM P (P8) for 7 and 14 days after transplantation (DAT). Low-P-tolerant cultivars increased root length by 31% and 119%, which was lower than the 62% and 144% increases in sensitive cultivars under P0 compared to P8 at 7 and 14 DAT, respectively. Acid phosphatase activity in low-P-tolerant cultivars exceeded that in sensitive cultivars by 5.2-fold and 2.0-fold at 7 and 14 DAT. Root exudates from each cultivar revealed 177 metabolites, with higher organic acid exudation in low-P-tolerant than sensitive cultivars under P0. Low-P-tolerant cultivars increased concentrations of specific metabolites (oxalate, GABA, quinate, citrate, AMP, 4-pyridoxate, and CMP), distinguishing them from low-P-sensitive cultivars under P0. The top five metabolomic pathways (purine metabolism, arginine and proline metabolism, TCA cycle, glyoxylate and dicarboxylate metabolism, alanine, aspartate, and glutamate metabolism) were more pronounced in low-P-tolerant cultivars at 14 DAT. These findings indicate that increasing root length was not an adaptation strategy under P deficiency; instead, tolerant cultivars exhibit enhanced root physiological traits, including increased acid phosphatase activity, organic acid exudation, specific metabolite release, and accelerated metabolic pathways under P deficiency.

The multifaceted nature of plant acid phosphatases: purification, biochemical features, and applications.

Acid phosphatases (EC 3.1.3.2) are the enzymes that catalyse transphosphorylation reactions and promotes the hydrolysis of numerous orthophosphate esters in acidic media, as a crucial element for the metabolism of phosphate in tissues. Inorganic phosphate (Pi) utilisation and scavenging, as well as the turnover of Pi-rich sources found in plant vacuoles, are major processes in which intracellular and secretory acid phosphatases function. Therefore, a thorough understanding of these enzymes' structural characteristics, specificity, and physiochemical properties is required to comprehend the function of acid phosphatases in plant energy metabolism. Furthermore, acid phosphatases are gaining increasing importance in industrial biotechnology due to their involvement in transphosphorylation processes and their ability to reduce phosphate levels in food products. Hence, this review aims to provide a comprehensive overview of the purification methods employed for isolating acid phosphatases from diverse plant sources, as well as their structural and functional properties. Additionally, the review explores the potential applications of these enzymes in various fields.

Acute thermal stress increased enzyme activity and muscle energy distribution of yellowfin tuna.

Heat is a powerful stressor for fish living in natural and artificial environments. Understanding the effects of heat stress on the physiological processes of fish is essential for better aquaculture and fisheries management. In this experiment, a heating rod was used to increase the temperature at 2°C/h to study the changes of energy allocation (CEA) and energy metabolity-related enzyme activities, including pepsin, trypsin, amylase, lipase, acid phosphatase, lactate dehydrogenase, alanine aminotransferase, glutamic oxalic aminotransferase and energy reserve (Ea), energy expenditure (ETS), in juvenile yellowfin tuna cells under acute temperature stress. The results showed that the Ea of juvenile yellowfin tuna muscles in response to high temperature (34°C) was significantly lower than that of the control (28°C), and it also increased ETS. At 6 h, CEA decreased slightly in the high-temperature group, but, the difference in CEA between 24 h and 0 h decreased. After heat stress for 6 h, the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and glutamic oxalacetic transaminase (AST) increased, indicating that the metabolic rate was accelerated. After heat stress for 24 h, the activity of ALT decreased, indicating that with time elapsed, the activities of some protein metabolizing enzymes increased, and some decreased. In this study, digestive enzymes, trypsin and lipase increased gradually. After heat stress, Ea and Ec change significantly. Yellowfin tuna muscles use lipids in response to sharp temperature increases at high temperatures, red muscles respond to temperature changes by increasing energy in the early stages, but not nearly as much, and white muscles reduce lipids.

Effect of phosphodiester composition in polyphosphoesters on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells.

Osteoporosis is a common bone disorder characterized by reduced bone density and increased risk of fractures. The modulation of bone cell functions, particularly the inhibition of osteoclastic differentiation, plays a crucial role in osteoporosis treatment. Polyphosphoesters (PPEs) have shown the potential in reducing the function of osteoclast cells, but the effect of their chemical structure on osteoclastic differentiation remains largely unexplored. In this study, we evaluated the effect of PPE's chemical structure on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells (BMNCs). PPEs containing phosphotriester and phosphodiester units at varying compositions were synthesized. Cytotoxicity testing confirmed the biocompatibility of the copolymers at concentrations below 0.5 mg/mL. Isolated from long bones, BMNCs were cultured in a differentiation medium supplemented with different PPE concentrations. Osteoclast formation was assessed through tartrate-resistant acid phosphatase and phalloidin staining. A significant decrease in the size of osteoclast cells formed upon BMNC contact with PPEs was observed, with a more pronounced effect observed at higher PPE concentrations. In addition, an increased composition of phosphodiester units in the PPEs yielded a decreased density of differentiated osteoclasts. Furthermore, real-time PCR analysis of major osteoclastic markers provided gene expression data that correlated with microscopic observations, confirming the effect of phosphodiester units in suppressing osteoclast differentiation of BMNCs from the early stages. These findings highlight the potential of PPEs as polymers are capable of modulating bone cell functions through their chemical structures.

In silico identification and analysis of potential inhibitors for acid phosphatase, HppA from Helicobacter pylori.

Helicobacter pylori is the most common cause of gastric ulcers and is associated with gastric cancer. The enzyme HppA of class C nonspecific acid phosphohydrolases (NSAPs) of H. pylori plays a crucial role in the electron transport chain. Herein, we report an in silico homology model of HppA consisting of a monomeric α + ß model. A high throughput structure-based virtual screening approach yielded potential inhibitors against HppA with higher binding energies. Further analyses of molecular interaction maps and protein-ligand fingerprints, followed by molecular mechanics-generalized Born surface area (MM-GBSA) end point binding energy calculations of docked complexes, resulted in the detection of top binders/ligands. Our investigations identified potential substrate-competitive small molecule inhibitors of HppA, with admissible pharmacokinetic properties. These molecules may provide a starting point for developing novel therapeutic agents against H. pylori.

Phosphorescence, fluorescence, and colorimetric triple-mode sensor for the detection of acid phosphatase and corresponding inhibitor.

Acid phosphatase (ACP) as a clinical diagnostic biomarker for several pathophysiological diseases has aroused widespread interest. Compared to commonly developed single-mode ACP detection technology, the multi-mode detection method with self-validation can provide more reliable results. Herein, we proposed a triple-mode phosphorescence, fluorescence, and colorimetric method for ACP detection in combination with CDs@SiO2. HAuCl4 with oxidase-like activity can catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to the blue oxide TMB (TMBox), offering absorption signals and quenching the phosphorescence and fluorescence of CDs@SiO2 based on the internal filtration effect (IFE). ACP can hydrolyze ascorbic acid 2-phosphate (AAP) to yield ascorbic acid (AA), thereby reducing TMBox to TMB, triggering solution fading and restoring phosphorescence and fluorescence signals. When the ACP inhibitor malathion is present, the reduction of TMBox is hindered, which successively led to the suppression of CDs@SiO2 phosphorescence and fluorescence signal recovery. According to these principles, triple-mode ACP (LOD = 0.0026 mU mL-1) and malathion detections (LOD = 0.039 µg mL-1) with favorable accuracy and sensitivity are realized. With simplicity, robustness, and versatility, the triple-mode sensor can be extended to the detection of the AAP hydrolase family and the screening of corresponding inhibitors.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

PAP(248-286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation.

The initial stage of fibril formation of C-terminal region PAP(248-286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248-286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation. The secondary nucleation includes interaction of protein monomers with the mature fibril surface that leads to protein spatial structural changes for further amyloid fibril formation. In this work, changes of the PAP(248-286) spatial structure were obtained during the secondary nucleation phase. Pulsed-field gradient (PFG) NMR was used to characterize the behavior of monomeric PAP(248-286) in water solution after PAP(248-286) seed addition. The self-diffusion coefficient showed compactization of the peptide monomer due to fibril-monomer interactions. PAP(248-286) spatial structural changes were detected with the help of high-resolution NMR spectroscopy and molecular dynamics (MD) simulation. The folding of PAP(248-286) occurs due to backbone chain bending in the region of H270 and T275 amino acid residues. Obtained folded conformation of PAP(248-286) emerging in the secondary nucleation process is energetically favorable and retains after monomer-amyloid interaction. The structural changes are associated with localization of PAP(248-286) hydrophobic surface regions, which are probably responsible for peptide monomer-amyloid interactions.