Enzyme Assay Research Articles

Higher serum uric acid as a risk factor for frailty in older adults: A nationwide population-based study.

Uric acid (UA), the terminal breakdown product of purine metabolism, possesses contradictory roles, functioning both as an inflammatory mediator and as an antioxidant. Its clinical relevance, particularly in geriatric populations, remains a topic of ongoing debate. Aiming to elucidate whether circulating UA is detrimental or beneficial to human health, we investigate the association between serum UA concentrations and the frailty index-a comprehensive measure of biological aging in a nationally representative cohort of community-dwelling older adults. We conducted a population-based, cross-sectional study utilizing data from the Korea National Health and Nutrition Examination Survey. The sample included 4268 participants aged 65years and above. A deficit accumulation frailty index (FI) was constructed using 38 items that assess physical, cognitive, psychological, and social domains. Based on the FI, participants were categorized into non-frail (FI≤0.15), pre-frail (0.15<FI≤0.25), or frail (FI>0.25). Serum UA levels were quantified through a colorimetric enzymatic assay. After controlling for confounders such as age, sex, socioeconomic status (including income and education level), lifestyle factors (smoking status), and medical history (hypertension, diabetes, dyslipidemia, stroke, cardiovascular diseases), and body mass index, serum UA levels were observed to be significantly higher in frail participants compared with their non-frail counterparts (P<0.001). Furthermore, serum UA concentrations demonstrated a positive correlation with the FI (P<0.001), and the odds ratio for frailty per 1mg/dL increase in serum UA was 1.22 (P<0.001). Additionally, older adults in the highest quartile of UA levels exhibited a significantly higher FI and 1.66-fold increased odds of frailty compared with those in the lowest quartile (P=0.011 and P=0.005, respectively). These findings suggest that elevated circulating UA levels may act as a pro-aging factor rather than an anti-aging one in older adults, highlighting its potential role in accelerating biological aging. The data further support the utility of serum UA as a potential blood-based biomarker for frailty in this demographic, contributing to the expanding evidence on its significance in geriatric health assessments.

Response of stream habitat and microbiomes to spruce budworm defoliation: New considerations for outbreak management.

Defoliation by eastern spruce budworm is one of the most important natural disturbances in Canadian boreal and hemi-boreal forests with annual area affected surpassing that of fire and harvest combined, and its impacts are projected to increase in frequency, severity, and range under future climate scenarios. Deciding on an active management strategy to control outbreaks and minimize broader economic, ecological, and social impacts is becoming increasingly important. These strategies differ in the degree to which defoliation is suppressed, but little is known about the downstream consequences of defoliation and, thus, the implications of management. Given the disproportionate role of headwater streams and their microbiomes on net riverine productivity across forested landscapes, we investigated the effects of defoliation by spruce budworm on headwater stream habitat and microbiome structure and function to inform management decisions. We experimentally manipulated a gradient of defoliation among 12 watersheds during a spruce budworm outbreak in the Gaspésie Peninsula, Québec, Canada. From May through October of 2019-2021, stream habitat (flow rates, dissolved organic matter [DOM], water chemistry, and nutrients), algal biomass, and water temperatures were assessed. Bacterial and fungal biofilm communities were examined by incubating six leaf packs for five weeks (mid-August to late September) in one stream reach per watershed. Microbiome community structure was determined using metabarcoding of 16S and ITS rRNA genes, and community functions were examined using extracellular enzyme assays, leaf litter decomposition rates, and taxonomic functional assignments. We found that cumulative defoliation was correlated with increased streamflow rates and temperatures, and more aromatic DOM (measured as specific ultraviolet absorbance at 254 nm), but was not correlated to nutrient concentrations. Cumulative defoliation was also associated with altered microbial community composition, an increase in carbohydrate biosynthesis, and a reduction in aromatic compound degradation, suggesting that microbes are shifting to the preferential use of simple carbohydrates rather than more complex aromatic compounds. These results demonstrate that high levels of defoliation can affect headwater stream microbiomes to the point of altering stream ecosystem productivity and carbon cycling potential, highlighting the importance of incorporating broader ecological processes into spruce budworm management decisions.

Folylpolyglutamate synthetase inactivation in relapsed ALL induces a druggable folate metabolic vulnerability

AimsThe antifolate methotrexate (MTX) is an anchor drug used in acute lymphoblastic leukemia (ALL) with poorly understood chemoresistance mechanisms in relapse. Herein we find decreased folate polyglutamylation network activities and inactivating FPGS mutations, both of which could induce MTX resistance and folate metabolic vulnerability in relapsed ALL. MethodsWe utilized integrated systems biology analysis of transcriptomic and genomic data from relapse ALL cohorts to infer hidden ALL relapse drivers and related genetic alternations during clonal evolution. The drug sensitivity assay was used to determine the impact of relapse-specific FPGS mutations on sensitivity to different antifolates and chemotherapeutics in ALL cells. We used liquid chromatography-mass spectrometry (LC-MS) to quantify MTX and folate polyglutamate levels in folylpoly-γ-glutamate synthetase (FPGS) mutant ALL cells. Enzymatic activity and protein degradation assays were also conducted to characterize the catalytic properties and protein stabilities of FPGS mutants. An ALL cell line-derived mouse leukemia xenograft model was used to evaluate the in vivo impact of FPGS inactivation on leukemogenesis and sensitivity to the polyglutamatable antifolate MTX as well as non-polyglutamatble lipophilic antifolate trimetrexate (TMQ). ResultsWe found a significant decrease in folate polyglutamylation network activities during ALL relapse using RNA-seq data. Supported by functional evidence, we identified multifactorial mechanisms of FPGS inactivation in relapsed ALL, including its decreased network activity and gene expression, focal gene deletion, impaired catalytic activity, and increased protein degradation. These deleterious FPGS alterations induce MTX resistance and inevitably cause marked intracellular folate shrinkage, which could be efficiently targeted by a polyglutamylation-independent lipophilic antifolate TMQ in vitro and in vivo. ConclusionsMTX resistance in relapsed ALL relies on FPGS inactivation, which inevitably induces a folate metabolic vulnerability, allowing for an efficacious antifolate ALL treatment strategy that is based upon TMQ, thereby surmounting chemoresistance in relapsed ALL.

Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

Developing Allosteric Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase.

The use of Fpocket and virtual screening techniques enabled us to identify potential allosteric druggable pockets within the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Of the compounds screened, compound 1 was identified as a promising inhibitor, lowering a SARS-CoV-2 RdRp activity to 57 % in an enzymatic assay at 10 μM concentration. The structure of compound 1 was subsequently optimized in order to preserve or enhance inhibitory activity. This involved the substitution of problematic ester and aromatic nitro groups with more inert functionalities. The N,N'-diphenylurea scaffold with two NH groups was identified as essential for the compound's activity but also exhibited high toxicity in Calu-3 cells. To address this issue, a scaffold hopping approach was employed to replace the urea core with potentially less toxic urea isosteres. This approach yielded several structural analogues with notable activity, specifically 2,2'-bisimidazol (in compound 55 with residual activity RA=42 %) and (1H-imidazol-2-yl)urea (in compounds 59 and 60, with RA=50 and 28 %, respectively). Despite these advances, toxicity remained a major concern. These compounds represent a promising starting point for further structure-activity relationship studies of allosteric inhibitors of SARS-CoV-2 RdRp, with the goal of reducing their cytotoxicity and improving aqueous solubility.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

Fragment-Guided Genome Mining of Octacyclic Cyclophane Alkaloids from Fungi.

Nature uses compact but functionalized biosynthetic fragments as building blocks to generate complex natural products. To leverage this strategy for the discovery of natural products with new scaffolds, we performed genome mining to identify biosynthetic gene clusters (BGCs) in fungi that embed genes that can synthesize targeted fragments. The three-enzyme pathway that biosynthesizes the strained dityrosine cyclophane in the herquline A pathway was used to identify a large number of potential BGCs that may use the cyclophane as a fragment. Characterization of a conserved BGC from fungal strains led to the isolation of octacyclin A, an octacyclic natural product with an unprecedented structure, including two hetero-[3.3.1]bicycles and a combination of fused, bridged, and macrocyclic rings. Biosynthetic steps leading to octacyclin A were fully elucidated using pathway reconstitution and enzymatic assays, unveiling intriguing chemical logic and new enzymatic reactions in building the octacyclic core. Our work demonstrates the potential utility of fragment-guided genome mining in expanding natural product chemical space.

Genome Mining and Genetic Manipulation Reveal New Isofuranonaphthoquinones in Nocardia Species.

The identification of specialized metabolites isolated from microorganisms is urgently needed to determine their roles in treating cancer and controlling multidrug-resistant pathogens. Naphthoquinones act as anticancer agents in various types of cancers, but some toxicity indicators have been limited in their appropriate application. In this context, new isofuranonaphthoquinones (ifnq) that are less toxic to humans could be promising lead compounds for developing anticancer drugs. The aim of this study is to identify and characterize novel furanonaphthoquinones (fnqs) from Nocardia sp. CS682 and to evaluate their potential therapeutic applications. Analysis of the genome of Nocardia sp. CS682 revealed the presence of a furanonaphthoquinone (fnq) gene cluster, which displays a similar genetic organization and high nucleotide sequence identity to the ifnq gene cluster from Streptomyces sp. RI-77, a producer of the naphthoquinones JBIR-76 and JBIR-77. In this study, the overexpression of the Streptomyces antibiotic regulatory protein (SARP) in Nocardia sp. CS682DR (nargenicin gene-deleted mutant) explicitly produced new fnqs, namely, NOC-IBR1 and NOC-IBR2. Subsequently, the role of the SARP regulator was confirmed by gene inactivation using CRISPR-Cas9 and complementation studies. Furthermore, antioxidant, antimicrobial, and cytotoxicity assays were performed for the isolated compounds, and it was found that NOC-IBR2 exhibited superior activities to NOC-IBR1. In addition, a flexible methyltransferase substrate, ThnM3, was found to be involved in terminal methylation of NOC-IBR1, which was confirmed by in vitro enzyme assays. Thus, this study supports the importance of genome mining and genome editing approaches for exploring new specialized metabolites in a rare actinomycete called Nocardia.

Evaluation of the antioxidant and antityrosinase activities of Prosopis juliflora fruit extract as a novel multifunctional bioactive ingredient and its potential applicability in pro-ageing and skin colour harmonization cosmetic products.

Prosopis juliflora, commonly known as algaroba or mesquite, was introduced and has since proliferated throughout the semi-arid region of the Caatinga biome. Various studies have documented its properties, including antimicrobial, antioxidant, and antitumor activities, attributed to the presence of diverse secondary metabolites such as alkaloids, terpenoids, tannins, and flavonoids. The objective of this study was to evaluate the antioxidant and antityrosinase activities of P. juliflora fruit extract as a multifunctional active ingredient, and to develop cosmetic formulations containing this vegetal extract for potential applications in skincare products targeting pro-ageing and skin colour homogenization properties. The extraction process followed established protocols. Chemical characterization of the extract involved quantification of total flavonoids and phenolic compounds, along with Liquid Chromatography-Mass Spectrometry (LC-MS) analysis. Invitro antioxidant activity was assessed using different methods. Antityrosinase activity was determined by employing enzymatic assays. Cosmetic formulations containing Disodium EDTA, Phenoxyethanol (and) Ethylhexyl Glycerin, Distilled Water, Sodium Acrylates Copolymer Lecithin, Polyacrylamide (and) C13-14 Isoparaffin (and) Laureth-7, and 3.0% of the investigated plant extract were subjected to preliminary and accelerated stability tests. The extract demonstrated a concentration of total flavonoids (1.71 ± 0.26 μg EQ/mg) and exhibited concentrations of phenolic compounds at 0.21 ± 0.01 mg EAG/g. Metabolites such as flavonoids and saponins were annotated, as well as some of their respective glycosidic derivatives. The extract showed antioxidant potential and the ability to inhibit the oxidation cascade in both the initiation and propagation phases. Moreover, the extract exhibited noteworthy inhibition of antityrosinase activity, presenting 62.48 ± 2.09 at a concentration of 30.00 mg/mL. The formulations were stable in accelerated stability tests over a 60-day period. This research not only demonstrates scientifically by demonstrating the potential of a plant from the Caatinga biome with antioxidant and antityrosinase properties in the development of cosmetic products aimed at pro-ageing effects and skin colour harmonization, but also adds value to the P. juliflora production chain. This valorization encompasses various aspects which include environmental, social, and biodiversity responsibilities.

Design and Synthesis of New Dihydropyrimidine Derivatives with a Cytotoxic Effect as Dual EGFR/VEGFR-2 Inhibitors.

We developed and synthesized tetrahydropyrimidine derivatives as possible cytotoxic agents to inhibit EGFR and VEGFR-2 in the present study. Our study completely assesses the cytotoxic efficiency of pyrimidine-based derivatives 4-15 against various cancer cell lines, revealing derivatives 12 and 15 for their remarkable activity with GI50 values of 37 and 35 nM, respectively, when compared to the reference erlotinib (33 nM). In vitro enzyme assays showed that target compounds, particularly 12, 14, and 15, effectively inhibited EGFR and VEGFR-2. In vitro enzyme testing revealed that compound 15 was the most promising, with IC50 values of 84 and 3.50 nM for EGFR and VEGFR-2, respectively. Additionally, an in vitro assessment of the novel targets' apoptotic potential revealed that both pro-apoptotic and antiapoptotic behaviors were promising, indicating that the apoptotic induction pathway is a strongly proposed action method for the newly developed targets. Finally, molecular docking experiments are elaborately discussed to corroborate the exact binding interactions of the most active hybrids 12 and 15 with the EGFR and VEGFR-2 active sites.

Deciphering the triclosan degradation mechanism in Sphingomonas sp. strain YL-JM2C: Implications for wastewater treatment and marine resources

Triclosan (TCS), an antimicrobial agent extensively incorporated into pharmaceuticals and personal care products, poses significant environmental risks because of its persistence and ecotoxicity. So far, a few microorganisms were suggested to degrade TCS, but the microbial degradation mechanism remains elusive. Here, a two-component angular dioxygenase (TcsAaAb) responsible for the initial TCS degradation was characterized in Sphingomonas sp. strain YL-JM2C. Whole-cell biotransformation and crude enzyme assays demonstrated that TcsAaAb catalyzed the conversion of TCS to 4-chlorocatechol and 3,5-dichlorocatechol rather than the commonly suggested product 2,4-dichlorophenol. Then two intermediates were catabolized by tcsCDEF cluster via an ortho-cleavage pathway. Critical residues (N262, F279, and F391) for substrate binding were identified via molecular docking and mutagenesis. Further, TcsAaAb showed activity toward methyl triclosan and nitrofen, suggesting its versatile potential for bioremediation. In addition, TCS-degrading genes were also present in diverse bacterial genomes in wastewater, ocean and soil, and a relatively high gene abundance was observed in marine metagenomes, revealing the transformation fate of TCS in environments and the microbial potential in pollutant removal. These findings extend the understanding of the microbe-mediated TCS degradation and contribute to the mining of TCS-degrading strains and enzymes, as well as their application in the bioremediation of contaminated environments.

Ultrasonic-driven chemical reduction synthesis of alizarin complexone-modified gold nanoparticles for dual-signal colorimetric and fluorometric sensing of histamine in seafood products

Alizarin complexone-modified gold nanoparticles (Au0-NPsALz) were synthesized using a proposed ultrasonic irradiation-assisted chemical reduction method. Ultrasonic irradiation powers, reaction time and alizarin complexone concentration had been proven to be the main parameters for controlling the nucleation and growth of Au0-NPsALz. In the synthesized ultrasonic irradiation-assisted chemical reduction conditions, Au0-NPsALz had a spherical oriented morphology with a uniform size of 17.84 ± 1.37 nm and are shiny red with a surface plasmon resonance (SPR) of 535 nm. A rapid colorimetric and fluorometric dual-mode detection strategy for selective detection of histamine in seafood was developed based on the self-assembly of Au0-NPsALz-Ni (II) complexes. Ni (II) can capture the histamine molecules close to Au0-NPsALz surfaces, making changes in the colorimetric and fluorometric responses of the solution. The quantitative analysis of histamine was realized through the variation of dual-signal colorimetric and fluorometric responses. Such Au0-NPsALz sensor offered good detection sensitivity for histamine with a detection limit (LOD) of 59.32 μmol L−1 and 116.20 μmol L−1 and wide linear response within the range of 10–10000 μmol L−1 (R2 = 0.9952) and 100–5000 μmol L−1 (R2 = 0.9947) for colorimetric and fluorometric measurement, respectively. Recoveries ranging from 94.99 to 103.29 % and 97.67–106.88 % for colorimetric and fluorometric assay were obtained, showing low levels of matrix effects. Particularly, the results of the dual-mode sensor were also validated by comparing with the HPLC method for improving the assay accuracy and dependability. Ultimately, the developed Au0-NPsALz colorimetric and fluorometric probe performs excellently in practical applications, with promising results for detecting histamine in seafood products.

Effects of Ethylenediaminetetraacetic Acid and Some Chlorides of Divalent Metals on the Initial Velocity of Crude Peroxidase from Watermelon Rind

Aim: This study investigated the effects of ethylenediaminetetraacetic acid (EDTA) and some chlorides of divalent metals on the initial velocity of crude peroxidase from watermelon rind Study design: In vitro enzyme assay. Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between September 2023 to November 2023. Methodology: The kinetics of the oxidation of Guaiacol by the crude peroxidase from the rind of watermelon in the presence of varying concentrations of EDTA and the chloride salts of iron, copper was determined spectrophotometrically by monitoring the oxidation of Guaiacol to produce a brown tetraguaiacol at a wavelength of 470nm. The various salt concentrations were varied between 0.5 mM and 3 mM. Each of the reaction mixtures used in the kinetic study comprised; 2.3 mL of 0.6 M sodium acetate buffer (pH 5.4), 0.2 mL of 0.02 mM Guaiacol, 0.1 mL of crude extract, 0.2 mL of varying concentration chloride salt, 0.2 mL of 2 mM of H2O2 added last to start the reaction. The final concentration of H2O2 in the 3 mL assay was 0.13 mM. The total volume of the reaction mixture was 3 mL. The absorbance was read every 2 seconds for one minute after adding hydrogen peroxide using a stop-clock. The control had no metal ion but replaced with distilled water Results: Results showed that EDTA reduced the activity of the enzyme in a concentration dependent manner. CuCl2 and FeCl2 activated the enzyme and FeCl2 being a better activator within the salt concentration range of 0.5 mM to 3 mM. Conclusion: These findings are of great importance to industries in understanding the mechanism of action of peroxidase from the rind of watermelon, especially as the search for cheap and alternative sources of peroxidases continues.

Identification of antimycobacterial 8-hydroxyquinoline derivatives as in vitro enzymatic inhibitors of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase

The increasing prevalence of drug-resistant Mycobacterium tuberculosis strains stimulates the discovery of new drug candidates. Among them are 8-hydroxyquinoline (8HQ) derivatives that exhibited antimicrobial properties. Unfortunately, there is a lack of data assessing possible targets for this class mainly against Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (MtInhA), a validated target in this field. Thus, the main purpose of this study was to identify 8HQ derivatives that are active against M. tuberculosis and MtInhA. Initially, the screening against the microorganism of a small antimicrobial library and its new derivatives that possess some structural similarity with MtInhA inhibitors identified four 7-substituted-8HQ (series 5 – 5a, 5c, 5d and 5i) and four 5-substituted-8HQ active derivatives (series 7 – 7a, 7c, 7d and 7j). In general, the 7-substituted 8-HQs were more potent and, in the enzymatic assay, were able to inhibit MtInhA at low micromolar range. However, the 5-substituted-8-HQs that presented antimycobacterial activity were not able to inhibit MtInhA. These findings indicate the non-promiscuous nature of 8-HQ derivatives and emphasize the significance of selecting appropriate substituents to achieve in vitro enzyme inhibition. Finally, 7-substituted-8HQ series are promising new derivatives for structure-based drug design and further development.

The gradual establishment of complex coumarin biosynthetic pathway in Apiaceae

Complex coumarins (CCs) represent characteristic metabolites found in Apiaceae plants, possessing significant medical value. Their essential functional role is likely as protectants against pathogens and regulators responding to environmental stimuli. Utilizing genomes and transcriptomes from 34 Apiaceae plants, including our recently sequenced Peucedanum praeruptorum, we conduct comprehensive phylogenetic analyses to reconstruct the detailed evolutionary process of the CC biosynthetic pathway in Apiaceae. Our results show that three key enzymes – p-coumaroyl CoA 2’-hydroxylase (C2’H), C-prenyltransferase (C-PT), and cyclase – originated successively at different evolutionary nodes within Apiaceae through various means of gene duplications: ectopic and tandem duplications. Neofunctionalization endows these enzymes with novel functions necessary for CC biosynthesis, thus completing the pathway. Candidate genes are cloned for heterologous expression and subjected to in vitro enzymatic assays to test our hypothesis regarding the origins of the key enzymes, and the results precisely validate our evolutionary inferences. Among the three enzymes, C-PTs are likely the primary determinant of the structural diversity of CCs (linear/angular), due to divergent activities evolved to target different positions (C-6 or C-8) of umbelliferone. A key amino acid variation (Ala161/Thr161) is identified and proven to play a crucial role in the alteration of enzymatic activity, possibly resulting in distinct binding forms between enzymes and substrates, thereby leading to different products. In conclusion, this study provides a detailed trajectory for the establishment and evolution of the CC biosynthetic pathway in Apiaceae. It explains why only a portion, not all, of Apiaceae plants can produce CCs and reveals the mechanisms of CC structural diversity among different Apiaceae plants.

Overexpression of the β-Subunit of Acid Ceramidase in the Epidermis of Mice Provokes Atopic Dermatitis-like Skin Symptoms.

We previously reported that a pathogenic abnormality in the barrier and water-holding functions of the stratum corneum (SC) in the skin of patients with atopic dermatitis (AD) is mainly attributable to significantly decreased levels of total ceramides in the SC. That decrease is mediated by the abnormal expression of a novel ceramide-reducing enzyme, sphingomyelin/glucosylceramide deacylase (SGDase), which is the β-subunit (ASAH1b) of acid ceramidase. In this study, we determined whether mice overexpressing ASAH1b in their epidermis develop AD-like skin symptoms. We generated transgenic (TG) mice overexpressing ASAH1b, regulated by the involucrin promoter, to localize its expression in the upper epidermis. After hair removal using a depilatory cream containing glycolic acid, the TG mice without any visible skin inflammation at 8 weeks of age had increased levels of ASAH1b and decreased levels of SC ceramide, with disrupted barrier functions measured by trans-epidermal water loss compared to the wild-type (WT) mice. Interestingly, enzymatic assays revealed that SGDase activity was not detectable in the skin of the TG mice compared to WT mice. Immunological staining revealed that there was an increased expression level of IL-33 in the epidermis and an accumulation of macrophages in the dermis of TG mice compared to WT mice, which are phenotypic characteristics of AD, that were exacerbated by tape-stripping of the skin. In the skin of the TG mice, the mRNA levels of IL-5, CCL11, IL-22, CXCL10, and IFNγ were significantly upregulated compared to the WT mice, and tape-stripping significantly increased the mRNA levels of IL-4, IL-33, CXCL1, CXCL12, TLR9, and CD163 compared to WT mice. These findings strongly indicate that the skin of the depilatory cream-treated TG mice exists in an atopic dry skin condition that is highly sensitive to various environmental stimuli. The sum of our results suggests that ASAH1b itself, even in the absence of its enzymatic activity, is a major etiologic factor for atopic dry skin symptoms via an unknown mechanism.

Penilumamide, a novel SIRT1 activator, protects UVB-induced photodamages in HaCaT cells

ABSTRACT Ultraviolet-B (UVB) radiation is a major physical factor that induces structural changes in human skin. The aim of this study was to determine whether the novel silent information regulator 1 (sirtuin 1 SIRT1) protein activator, penilumamide, exerted any protective effects against UVB-induced skin damage using human HaCaT keratinocytes as a model. Enzymatic assays were performed to determine the SIRT1-activating ability of penilumamide, which was compared with that of resveratrol, a potent natural product SIRT1 activator with antioxidant and anti-inflammatory properties. Penilumamide markedly activated SIRT1 enzyme activity compared to resveratrol. To further investigate the protective effect of penilumamide against UVB-induced cytotoxicity, HaCaT cells were pretreated with penilumamide (10 μM) for 24 hr followed by irradiation with UVB (40 mJ/cm2). UVB (40 mJ/cm2) irradiation significantly reduced cell viability in a time-dependent manner, whereas pretreatment with penilumamide blocked this effect. Further, penilumamide decreased the levels of intracellular reactive oxygen species (ROS) generated by UVB irradiation in HaCaT cells. Pretreatment with penilumamide also prevented UVB irradiation-induced changes in mitochondrial membrane potential (ΔΨ m). In addition, pretreatment with penilumamide significantly reduced the expression levels of pro-inflammatory cytokines, interleukin (IL)-6, IL-8, and IL-10 and phosphorylation of nuclear factor-kB (NF-kB). These results indicate that penilumamide protects HaCaT cells from UVB-induced inflammation. Taken together data demonstrate that penilumamide exerted protective effects against UVB-induced ROS generation in HaCaT cells. Therefore, penilumamide may be considered to be used as a new SIRT1 activator to protect human keratinocyte against UVB-induced damage.

The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O2-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

Deficiency of the sphingosine-1-phosphate (S1P) transporter Mfsd2b protects the heart against hypertension-induced cardiac remodeling by suppressing the L-type-Ca2+ channel.

The erythrocyte S1P transporter Mfsd2b is also expressed in the heart. We hypothesized that S1P transport by Mfsd2b is involved in cardiac function. Hypertension-induced cardiac remodeling was induced by 4-weeks Angiotensin II (AngII) administration and assessed by echocardiography. Ca2+ transients and sarcomere shortening were examined in adult cardiomyocytes (ACM) from Mfsd2b+/+ and Mfsd2b-/- mice. Tension and force development were measured in skinned cardiac fibers. Myocardial gene expression was determined by real-time PCR, Protein Phosphatase 2A (PP2A) by enzymatic assay, and S1P by LC/MS, respectively. Msfd2b was expressed in the murine and human heart, and its deficiency led to higher cardiac S1P. Mfsd2b-/- mice had regular basal cardiac function but were protected against AngII-induced deterioration of left-ventricular function as evidenced by ~ 30% better stroke volume and cardiac index, and preserved ejection fraction despite similar increases in blood pressure. Mfsd2b-/- ACM exhibited attenuated Ca2+ mobilization in response to isoprenaline whereas contractility was unchanged. Mfsd2b-/- ACM showed no changes in proteins responsible for Ca2+ homeostasis, and skinned cardiac fibers exhibited reduced passive tension generation with preserved contractility. Verapamil abolished the differences in Ca2+ mobilization between Mfsd2b+/+ and Mfsd2b-/- ACM suggesting that S1P inhibits L-type-Ca2+ channels (LTCC). In agreement, intracellular S1P activated the inhibitory LTCC phosphatase PP2A in ACM and PP2A activity was increased in Mfsd2b-/- hearts. We suggest that myocardial S1P protects from hypertension-induced left-ventricular remodeling by inhibiting LTCC through PP2A activation. Pharmacologic inhibition of Mfsd2b may thus offer a novel approach to heart failure.

Genome-Wide Isolation of VIN Gene Family and Functional Identification of HpVIN4 in Red Pitaya (Hylocereus polyrhizus)

Soluble sugars, including glucose, fructose and sucrose, are the most important determinants that affect the flavor and quality of red pitaya (Hylocereus polyrhizus) fruit. Vacuolar invertase (VIN), which catalyzes sucrose hydrolysis into glucose and fructose, is a key type of enzyme responsible for soluble sugar metabolism in plant growth and development. Herein, we conducted genome-wide identification, gene expression analysis, subcellular localization and an enzymatic properties assay for the VIN-encoding genes from red pitaya. During red pitaya fruit development towards ripening, the enzymatic activities of VIN showed an up-regulated trend towards ripening. In total, four isoforms (HpVIN1–4) of the VIN-encoding gene were identified from the pitaya genome. Sequence alignment results revealed that the HpVIN1, HpVIN3 and HpVIN4 proteins contained essential motifs for targeting the vacuole and conserved motifs or residues responsible for sucrose binding and hydrolysis. Gene expression pattern analyses revealed that the level of HpVIN4 was obviously increasing during fruit development and acted as the most abundant VIN isoform towards ripening. Subcellular localization detection via transient expression in Arabidopsis thaliana mesophyll protoplasts revealed that the HpVIN4 protein was localized in the vacuole. Growth complementation tests of heterologous expression in the invertase-deficient baker’s yeast strain suggested that the HpVIN4 protein had a sucrose hydrolysis activity and could restore the yeast growth in vivo. The identification of enzymatic properties in vitro demonstrated that the HpVIN4 protein could degrade sucrose into glucose and fructose with an optimum pH of 4.0. Specifically, the HpVIN4 protein had an estimated Km value of 5.15 ± 1.03 mmol·L−1 for sucrose hydrolysis. Ultimately, this study provides a comprehensive understanding of the potential roles of VINs during fruit development and towards ripening and provides functional gene resources for regulating soluble sugar accumulation in red pitaya fruit.