Sensitive detection of choline and nicotine in real samples by switching upconversion luminescence.

Nicotine (3-(1-methyl-2-pyrrolidinyl)pyridine) is one of the most common addictive substances, causing the trace detection of nicotine to be very necessary. Herein, we designed and prepared a functionalized nanocomposite CS-PAA (NaYF4:19.5%Yb,0.5%Tm@NaYF4-PAA) using a simple method. The nicotine concentration was quantitatively detected through the inhibition of choline oxidase activity by nicotine and the luminescence intensity of CS-PAA being quenched by Fe3+. The mechanism of Fe3+ quenching CS-PAA emission was inferred by luminescence lifetime and UV-vis absorption spectra characterization. During the nicotine detection, both excitation (980 nm) and emission (802 nm) wavelengths of CS-PAA enable the avoidance of the interference of background fluorescence in complicated food objects, thus providing high selectivity and sensitivity with a linear range of 5-750 ng/mL and a limit of detection of 9.3 nM. The method exhibits an excellent recovery and relative standard deviation, indicating high accuracy and repeatability of the detection of nicotine.

Master corepressor inactivation through multivalent SLiM-induced polymerization mediated by the oncogene suppressor RAI2.

While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.

Chemoenzymatic Synthesis of 2-Aryl Thiazolines from 4-Hydroxybenzaldehydes Using Vanillyl Alcohol Oxidases.

Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis.

Characterization of subunit interactions in the hetero-oligomeric retinoid oxidoreductase complex.

The hetero-oligomeric retinoid oxidoreductase complex (ROC) catalyzes the interconversion of all-trans-retinol and all-trans-retinaldehyde to maintain the steady-state output of retinaldehyde, the precursor of all-trans-retinoic acid that regulates the transcription of numerous genes. The interconversion is catalyzed by two distinct components of the ROC: the NAD(H)-dependent retinol dehydrogenase 10 (RDH10) and the NADP(H)-dependent dehydrogenase reductase 3 (DHRS3). The binding between RDH10 and DHRS3 subunits in the ROC results in mutual activation of the subunits. The molecular basis for their activation is currently unknown. Here, we applied site-directed mutagenesis to investigate the roles of amino acid residues previously implied in subunit interactions in other SDRs to obtain the first insight into the subunit interactions in the ROC. The results of these studies suggest that the cofactor binding to RDH10 subunit is critical for the activation of DHRS3 subunit and vice versa. The C-terminal residues 317-331 of RDH10 are critical for the activity of RDH10 homo-oligomers but not for the binding to DHRS3. The C-terminal residues 291-295 are required for DHRS3 subunit activity of the ROC. The highly conserved C-terminal cysteines appear to be involved in inter-subunit communications, affecting the affinity of the cofactor binding site in RDH10 homo-oligomers as well as in the ROC. Modeling of the ROC quaternary structure based on other known structures of SDRs suggests that its integral membrane-associated subunits may be inserted in adjacent membranes of the endoplasmic reticulum (ER), making the formation and function of the ROC dependent on the dynamic nature of the tubular ER network.

A Standalone ß-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold.

Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone ß-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone ß-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone ß-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.

Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis.

Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.

Membrane binding properties of the C-terminal segment of retinol dehydrogenase 8.

Light absorption by rhodopsin leads to the release of all-trans retinal (ATRal) in the lipid phase of photoreceptor disc membranes. Retinol dehydrogenase 8 (RDH8) then reduces ATRal into all-trans retinol, which is the first step of the visual cycle. The membrane binding of RDH8 has been postulated to be mediated by one or more palmitoylated cysteines located in its C-terminus. Different peptide variants of the C-terminus of RDH8 were thus used to obtain information on the mechanism of membrane binding of this enzyme. Steady-state and time-resolved fluorescence measurements were performed using short and long C-terminal segments of bovine RDH8, comprising one or two tryptophan residues. The data demonstrate that the amphipathic alpha helical structure of the first portion of the C-terminus of RDH8 strongly contributes to its membrane binding, which is also favored by palmitoylation of at least one of the cysteines located in the last portion of the C-terminus.

NAD(H) phosphates mediate tetramer assembly of human C-terminal binding protein (CtBP).

C-terminal binding proteins (CtBPs) are cotranscriptional factors that play key roles in cell fate. We have previously shown that NAD(H) promotes the assembly of similar tetramers from either human CtBP1 and CtBP2 and that CtBP2 tetramer destabilizing mutants are defective for oncogenic activity. To assist structure-based design efforts for compounds that disrupt CtBP tetramerization, it is essential to understand how NAD(H) triggers tetramer assembly. Here, we investigate the moieties within NAD(H) that are responsible for triggering tetramer formation. Using multiangle light scattering (MALS), we show that ADP is able to promote tetramer formation of both CtBP1 and CtBP2, whereas AMP promotes tetramer assembly of CtBP1, but not CtBP2. Other NAD(H) moieties that lack the adenosine phosphate, including adenosine and those incorporating nicotinamide, all fail to promote tetramer assembly. Our crystal structures of CtBP1 with AMP reveal participation of the adenosine phosphate in the tetrameric interface, pinpointing its central role in NAD(H)-linked assembly. CtBP1 and CtBP2 have overlapping but unique roles, suggesting that a detailed understanding of their unique structural properties might have utility in the design of paralog-specific inhibitors. We investigated the different responses to AMP through a series of site-directed mutants at 13 positions. These mutations reveal a central role for a hinge segment, which we term the 120s hinge that connects the substrate with coenzyme-binding domains and influences nucleotide binding and tetramer assembly. Our results provide insight into suitable pockets to explore in structure-based drug design to interfere with cotranscriptional activity of CtBP in cancer.

Cryo-EM structure of CtBP2 confirms tetrameric architecture.

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis, and metastasis. NADH-dependent CtBP activation has been implicated in multiple types of cancer and poor patient prognosis. Central to understanding activation of CtBP in oncogenesis is uncovering how NADH triggers protein assembly, what level of assembly occurs, and if oncogenic activity depends upon such assembly. Here, we present the cryoelectron microscopic structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is tetrameric. The physiological relevance of the observed tetramer was demonstrated in cell culture, showing that CtBP tetramer-destabilizing mutants are defective for cell migration, transcriptional repression of E-cadherin, and activation of TIAM1. Together with our cryoelectron microscopy studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.

Design, synthesis and evaluation of covalent inhibitors of DprE1 as antitubercular agents.

Decaprenylphosphoryl-ß-d-ribose 2'-oxidoreductase (DprE1) is a promising drug target for the development of novel anti-tubercular agents, and inhibitors of DprE1 are being investigated extensively. Among them, the 1,3-benzothiazinone compounds such as BTZ043, and its closer congener, PBTZ169, are undergoing clinical studies. It has been shown that both BTZ compounds are prodrugs, the nitro group is reduced to nitroso first, to which an adjacent Cys387 in the DprE1 binding pocket is covalently bound and results in suicide enzyme inhibition. We figured that replacement of the nitro with an electrophilic warhead would still achieve covalent interaction with nucleophilic Cys387, while the required reductive activation could be circumvented. To test this hypothesis, a number of covalent inhibitors of DprE1 were designed and prepared. The compounds inhibitory potency against DprE1 and anti-tubercular activity were investigated, their chemical reactivity, formation of covalent adduct between the warhead and the enzyme was demonstrated by mass spectrometry.

An Unprecedented Ring-Contraction Mechanism in Cobalamin-Dependent Radical S-Adenosylmethionine Enzymes.

A unique member of the family of cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes, OxsB, catalyzes the ring constriction of deoxyadenosine triphosphate (dATP) to the base oxetane aldehyde phosphate, a crucial precursor for oxetanocin A (OXT-A), which is an antitumor, antiviral, and antibacterial compound. This enzyme reveals a new catalytic function for this big family that is different from the common methylation. On the basis of density functional theory calculations, a mechanism has been proposed to mainly include that the generation of 5'-deoxyadenosine radical, a hydrogen transfer forming 2'-dATP radical, and a Cbl-catalyzed ring contraction of the deoxyribose in 2'-dATP radical. The ring contraction is a concerted rearrangement step accompanied by an electron transfer from the deoxyribose hydroxyl oxygen to CoIII without any ring-opening intermediate. CoIICbl has been ruled out as an active state. Other mechanistic characteristics are also revealed. This unprecedented non-methylation mechanism provides a new catalytic repertoire for the family of radical SAM enzymes, representing a new class of ring-contraction enzymes.

Stabilization of C-terminal binding protein 2 by cellular inhibitor of apoptosis protein 1 via BIR domains without E3 ligase activity.

C-terminal binding protein 2 (CtBP2) is a transcriptional co-repressor that regulates many genes involved in normal cellular events. Because CtBP2 overexpression has been implicated in various human cancers, its protein levels must be precisely regulated. Previously, we reported that CtBP1 and CtBP1-mediated transcriptional repression are regulated by X-linked inhibitor of apoptosis protein (XIAP). In the present study, we sought to investigate whether CtBP2 is also regulated by XIAP or any other human IAP. We found that cIAP1 interacts with CtBP2 via through BIR domains to regulates the steady-state levels of CtBP2 protein in the nucleus. The levels of CtBP2 were gradually increased upon cIAP1 overexpression and downregulated upon cIAP1 depletion. Interestingly, the RING domain of cIAP1 responsible for E3 ligase activity was not required for this regulation. Finally, the levels of CtBP2 modulated by cIAP1 affected the transcription of CtBP2 target genes and subsequent cell migration. Taken together, our data demonstrate a novel function of cIAP1 which involves protecting CtBP2 from degradation to stabilize its steady-state level. These results suggest that cIAP1 might be a useful target in strategies aiming to downregulate the steady-state level of CtBP2 protein in treating human cancers.

Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1.

In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The Km values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414 mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.

Assay of Phospholipase D Activity by an Amperometric Choline Oxidase Biosensor.

A novel electrochemical method to assay phospholipase D (PLD) activity is proposed based on the employment of a choline biosensor realized by immobilizing choline oxidase through co-crosslinking on an overoxidized polypyrrole film previously deposited on a platinum electrode. To perform the assay, an aliquot of a PLD standard solution is typically added to borate buffer containing phosphatidylcholine at a certain concentration and the oxidation current of hydrogen peroxide is then measured at the rotating modified electrode by applying a detection potential of + 0.7 V vs. SCE. Various experimental parameters influencing the assay were studied and optimized. The employment of 0.75% (v/v) Triton X-100, 0.2 mM calcium chloride, 5 mM phosphatidylcholine, and borate buffer at pH 8.0, ionic strength (I) 0.05 M allowed to achieve considerable current responses. In order to assure a controlled mass transport and, at the same time, high sensitivity, an electrode rotation rate of 200 rpm was selected. The proposed method showed a sensitivity of 24 (nA/s)(IU/mL)-1, a wide linear range up to 0.33 IU/mL, fast response time and appreciable long-term stability. The limit of detection, evaluated from the linear calibration curve, was 0.005 IU/mL (S/N = 3). Finally, due to the presence of overoxidized polypyrrole film characterized by notable rejection properties towards electroactive compounds, a practical application to real sample analysis can be envisaged.

Enhancing hydrogel-based long-lasting chemiluminescence by a platinum-metal organic framework and its application in array detection of pesticides and d-amino acids.

Organophosphorus pesticides (OPs) are harmful to people's health and d-amino acids (d-AAs) in the human body are closely related to various diseases. So, detection of OPs in foods and d-AAs in serum is important for food safety and clinical diagnosis. Herein, a long-lasting chemiluminescence (CL) imaging sensor was constructed for the detection of OPs and d-AAs. The method was based on N-(4-aminobutyl)-N-ethylisoluminol/Co2+/chitosan (ABEI/Co2+/CS) hydrogels, where metal organic framework materials (MOF-Pt) were selected as catalysts to improve the sensitivity greatly. Under the catalysis of acetylcholinesterase (AChE) and choline oxidase (CHO), H2O2 was produced by using acetylcholine chloride (ACh) as a substrate, which was sensitive to the proposed CL system. OPs inhibited the activity of AChE and decreased the production of H2O2, reducing CL intensity. The linear range of the method for chlorpyrifos was 0.5 ng mL-1-1.0 µg mL-1, with a limit of detection (LOD) of 0.21 ng mL-1. Seventeen kinds of OPs can be visually and simultaneously discerned by the CL imager. On the other hand, d-AAs were catalyzed and oxidized by d-α-amino oxidase (DAAO) to produce H2O2. Thus, d-Ala in serum was used as a model to be detected by the proposed method. The linear range for d-Ala was 1.0 µM-10 mM, with an LOD of 0.12 µM.

Dual-enzymatically crosslinked hyaluronic acid hydrogel as a long-time 3D stem cell culture system.

Stem cell-based tissue engineering shows enormous potential for regenerative medicine. Three-dimensional (3D) stem cell culture is the most basic aspect of tissue engineering. However, achievement of a perfect scaffold for highly efficient 3D cell culture is currently still limited. Herein, a new hyaluronic acid hydrogel dual-enzymatically crosslinked by horseradish peroxidase and choline oxidase is developed as a 3D stem cell culture system. This hydrogel possesses superior stability over two months, controllable biodegradability with hyaluronidases, a high swelling ratio exceeding 6000%, and excellent cytocompatibility in vitro and biocompatibility in vivo. More importantly, a long-time and highly cellular activity 3D culture of bone marrow-derived mesenchymal stem cells was achieved in vitro over 20 days. All these encouraging results highlight the great potential of this new hydrogel for 3D culture and tissue engineering.

Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group.

Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed.

Transcriptional co-repressor CtBP2 orchestrates epithelial-mesenchymal transition through a novel transcriptional holocomplex with OCT1.

The epithelial to mesenchymal transition (EMT) is a cell intrinsic program controlling cellular morphological and phenotypic remodeling in a wide range of biological processes. Despite the accumulating evidence, the transcriptional networks regulating EMT still remain to be elucidated. In this study, we demonstrate that C-terminal binding protein 2 (CtBP2), a critical transcriptional co-repressor harboring pyridine nucleotide sensing capability, orchestrates the EMT program at least in part through a novel transcriptional interaction with an octamer transcription factor, OCT1 (POU2F1, POU class 2 homeobox 1). We identified novel interactions of CtBP2 with several octamer transcription factors, and CtBP2 exhibits a direct interaction with OCT1 in particular. OCT1 accelerates the EMT program as reported, which is diminished by the mutation of the CtBP-binding motif in OCT1, suggesting OCT1 represses epithelial gene expression through recruiting the co-repressor CtBP2. In accordance with these findings, a canonical EMT activator transforming growth factor-ß (TGF-ß) promotes the formation of the CtBP2/OCT1 complex. Our observations illustrate the role of CtBP2 to orchestrate the EMT program through the interaction with OCT1 and highlight the potential of therapeutic exploitation of this new transcriptional system for a wide range of diseases.

Development of choline biosensor using toluidine blue O as mediator.

A new choline oxidase (ChO) and toluidine blue O (TBO) based amperometric choline biosensor was reported in this article. An amperometric choline biosensor with immobilization of TBO (as a mediator), ChO onto polypyrrole-polyvinylsulphonate (PPy-PVS) film was accomplished on the surface of a platinum electrode. ChO was immobilized on PPy-PVS film by cross-linking with glutaraldehyde (GA). TBO was used as the mediator. Choline is oxidized to betaine and hydrogen peroxide in an oxygenated environment by ChO. Mediator reduced by reaction with hydrogen peroxide. The amperometric response was based upon the electrocatalytic properties of TBO. Optimum pH and temperature values were 7.0 and 30 °C, respectively. There was linearity between 1.0 × 10-8 and 2.0 × 10-8 M (R2 = 0.9805). The detection limit of the biosensor was 1.0 × 10-9 M and response time of the biosensor was 200 s. The storage stability and reproducibility of the biosensor were also investigated. Interfering effect of several interferants such as ascorbic acid, uric acid, alanine, dopamine, paracetamol, cysteine, and glucose on the choline biosensor was examined. The developed biosensor was tested in determinations of content in a synthetic blood sample.

Retinol dehydrogenase 12 (RDH12): Role in vision, retinal disease and future perspectives.

Retinol dehydrogenase 12 (RDH12) is an NADPH-dependent retinal reductase, which is expressed in the inner segments of the photoreceptors. It functions as part of the visual cycle, which is a series of enzymatic reactions required for the regeneration of the visual pigment, and has also been implicated in detoxification of lipid peroxidation products. Mutations in RDH12 have been linked to Leber congenital amaurosis (LCA) and autosomal dominant retinitis pigmentosa. A number of in-vitro studies have shown that mutations in RDH12 result in little or no enzyme activity. Knockout mouse models however do not recapitulate the severe phenotype observed in patients, resulting in a limited understanding of the disease mechanisms. With gene replacement and small molecule drugs emerging for inherited retinal dystrophies, herein we provide a review of RDH12 structure, its role in vision and the current understanding of disease mechanisms linked to clinical phenotype to support therapeutic development.