A 3D-Predicted Structure of the Amine Oxidase Domain of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has been recognized as an attractive drug target for anti-fibrotic and anti-tumor therapies. However, the structure-based drug design of LOXL2 has been very challenging due to the lack of structural information of the catalytically-competent LOXL2. In this study; we generated a 3D-predicted structure of the C-terminal amine oxidase domain of LOXL2 containing the lysine tyrosylquinone (LTQ) cofactor from the 2.4Å crystal structure of the Zn2+-bound precursor (lacking LTQ; PDB:5ZE3); this was achieved by molecular modeling and molecular dynamics simulation based on our solution studies of a mature LOXL2 that is inhibited by 2-hydrazinopyridine. The overall structures of the 3D-modeled mature LOXL2 and the Zn2+-bound precursor are very similar (RMSD = 1.070Å), and disulfide bonds are conserved. The major difference of the mature and the precursor LOXL2 is the secondary structure of the pentapeptide (His652-Lys653-Ala654-Ser655-Phe656) containing Lys653 (the precursor residue of the LTQ cofactor). We anticipate that this peptide is flexible in solution to accommodate the conformation that enables the LTQ cofactor formation as opposed to the ß-sheet observed in 5ZE3. We discuss the active site environment surrounding LTQ and Cu2+ of the 3D-predicted structure.

In silico characterization of fructosyl peptide oxidase properties from Eupenicillium terrenum.

Fructosyl peptide oxidase (FPOX) enzyme from Eupenicillium terrenum has a high potential to be applied as a diagnostic enzyme. The aim of the present study is the characterization of FPOX from E. terrenum using different bioinformatics tools. The computational prediction of the RNA and protein secondary structures of FPOX, solubility profile in Escherichia coli, stability, domains, and functional properties were performed. In the FPOX protein, six motifs were detected. The d-amino acid oxidase motif was found as the most important motif that is a FAD-dependent oxidoreductase. The cysteines including 97, 154, 234, 280, and 360 showed a lower score than -10 that have a low possibility for participitation in the formation of the SS bond. The 56.52% of FPOX amino acids are nonpolar. Random coils are dominant in the FPOX sequence, followed by alpha-helix and extended strand. The fpox gene is capable of generating a stable RNA secondary structure (-423.90 kcal/mol) in E. coli. FPOX has a large number of hydrophobic amino acids. FPOX showed a low solubility in E. coli which has several aggregation-prone sites in its 3-D structure. According to the scores, the best mutation candidate for increasing solubility was the conversion of methionine 302 to arginine. The melting temperature of FPOX based on its amino acid sequence was 55°C to 65°C. The amounts of thermodynamic parameters for the FPOX enzyme were -137.4 kcal/mol, -3.59 kcal/(mol K), and -6.8 kcal/mol for standard folding enthalpy, heat capacity, and folding free energy, respectively. In conclusion, the in silico study of proteins can provide a valuable method for better understanding the protein properties and functions for use in our purposes.

Oligomeric States and Hydrodynamic Properties of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution.

Accurate glutamate monitoring in foodstuffs by a sensitive and interference-free glutamate oxidase based disposable amperometric biosensor.

L-Glutamate (L-Glu) is a well-known flavour enhancer that is present in several foodstuffs. Although L-Glu is generally recognized as safe, the use in foodstuffs remains controversial and then its fast and accurate monitoring represents an important issue. In this work a sensitive and interference-free disposable amperometric biosensor for glutamate monitoring in foodstuffs was developed. The biosensor was prepared by immobilizing glutamate oxidase through co-crosslinking with bovine serum albumin and glutaraldehyde onto a screen printed disposable platinum electrode modified with a permselective overoxidized polypyrrole film. The enzyme immobilization was optimized by using different experimental procedures. The optimized glutamate biosensor was integrated in a flow injection system and characterized in terms of linearity (0.005-1.0 mM, r2 = 0.992), limits of detection (1.8 µM) and quantitation (5.4 µM), repeatability (RSD < 3%) and stability of response under operational conditions (up to 50 h, over 400 analysis). The biosensor showed also excellent anti-interference characteristics towards the main electroactive interferents present in food matrices, and this allowed the application to the accurate monitoring of glutamate in different foodstuffs.

Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159.

D-amino acid production from 2-keto acid by reductive amination is an attractive pathway because of its high yield and environmental safety. StDAPDH, a meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum, was the first meso-DAPDH to show amination of 2-keto acids. Furthermore, StDAPDH shows excellent thermostability compared to other meso-DAPDHs. However, the cofactor of StDAPDH is NADP(H), which is less common than NAD(H) in industrial applications. Therefore, cofactor engineering for StDAPDH is needed. In this study, the highly conserved cofactor binding sites around the adenosine moiety of NADPH were targeted to determine cofactor specificity. Lysine residues within a loop were found to be critical for the cofactor specificity of StDAPDH. Replacement of lysine with arginine resulted in the activity of pyruvic acid with NADH as the cofactor. The affinity of K159R to pyruvic acid was equal with NADH or NADPH as the cofactor, regardless of the mutation. Molecular dynamics simulations revealed that the large steric hindrance of arginine and the interaction of the salt bridge between NADH and arginine may have restricted the free movement of NADH, which prompted the formation of a stable active conformation of mutant K159R. These results provide further understanding of the catalytic mechanism of StDAPDH and guidance for the cofactor engineering of StDAPDH.

Electroenzymatic glutamate sensing at near the theoretical performance limit.

The sensitivity and response time of glutamate sensors based on glutamate oxidase immobilized on planar platinum microelectrodes have been improved to near the theoretical performance limits predicted by a detailed mathematical model. Microprobes with an array of electroenzymatic sensing sites have emerged as useful tools for the monitoring of glutamate and other neurotransmitters in vivo; and implemented as such, they can be used to study many complex neurological diseases and disorders including Parkinson's disease and drug addiction. However, less than optimal sensitivity and response time has limited the spatiotemporal resolution of these promising research tools. A mathematical model has guided systematic improvement of an electroenzymatic glutamate microsensor constructed with a 1-2 µm-thick crosslinked glutamate oxidase layer and underlying permselective coating of polyphenylenediamine and Nafion reduced to less than 200 nm thick. These design modifications led to a nearly 6-fold improvement in sensitivity to 320 ± 20 nA µM-1 cm-2 at 37 °C and a ∼10-fold reduction in response time to 80 ± 10 ms. Importantly, the sensitivity and response times were attained while maintaining a low detection limit and excellent selectivity. Direct measurement of the transport properties of the enzyme and polymer layers used to create the biosensors enabled improvement of the mathematical model as well. Subsequent model simulations indicated that the performance characteristics achieved with the optimized biosensors approach the theoretical limits predicted for devices of this construction. Such high-performance glutamate biosensors will be more effective in vivo at a size closer to cellular dimension and will enable better correlation of glutamate signaling events with electrical recordings.

An amperometric biosensor for specific detection of glycated hemoglobin based on recombinant engineered fructosyl peptide oxidase.

Here, we present a specific biosensor based on the detection of glycated hemoglobin (HbA1c) proteolytic digestion product, fructosyl valyl histidine (Fru-ValHis). A recombinant engineered fructosyl peptide oxidase (FPOX) enzyme with improved specificity was immobilized on the electrode surface modified by chitosan (CHIT), graphene oxide (GO) and gold nanoparticles (AuNPs). The biosensor exhibited a linear response toward different concentrations of Fru-ValHis ranging from 0.1 to 2 mM with a sensitivity of 8.45 µA mM-1 cm-2. Detection limit of the current biosensor for Fru-ValHis was 0.3 µM as the lowest quantity required giving a signal to a background. Analytical recovery of added Fru-ValHis in whole blood was 95.1-98.35% for FPOX/AuNPs/GO/CHIT/FTO electrode. For Fru-ValHis determination by FPOX-AuNPs-GO-CHIT/FTO electrode, within-run coefficient of variation (CV) was between 1.3% and 2.4% and between run CV was between 2.1% and 3.5%. A significant change in electron transfer resistance after the incubation of FPOX-modified electrode with Fru-ValHis was observed, while no response was achieved with control, indicating specific measurement of Fru-ValHis. Moreover, designed biosensor measured HbA1c in human blood samples and the results were well agreed with that obtained with NORUDIA™ N HbA1c diagnostic kit. Overall, suitable specificity of the engineered FPOX made the bioelectrode responded well to the Fru-ValHis level, which demonstrates a promising application for specific detection of HbA1c biomarker.

Scavenger Receptor Cysteine-Rich domains of Lysyl Oxidase-Like2 regulate endothelial ECM and angiogenesis through non-catalytic scaffolding mechanisms.

Lysyl oxidases are major actors of microenvironment and extracellular matrix (ECM) remodeling. These cross-linking enzymes are thus involved in many aspects of physiopathology, including tumor progression, fibrosis and cardiovascular diseases. We have already shown that Lysyl Oxidase-Like 2 (LOXL2) regulates collagen IV deposition by endothelial cells and angiogenesis. We here provide evidence that LOXL2 also affects deposition of other ECM components, including fibronectin, thus altering structural and mechanical properties of the matrix generated by endothelial cells. LOXL2 interacts intracellularly and directly with collagen IV and fibronectin before incorporation into ECM fibrillar structures upon exocytosis, as demonstrated by TIRF time-lapse microscopy. Furthermore, surface plasmon resonance experiments using recombinant scavenger receptor cysteine-rich (SRCR) domains truncated for the catalytic domain demonstrated their direct binding to collagen IV. We thus used directed mutagenesis to investigate the role of LOXL2 catalytic domain. Neither enzyme activity nor catalytic domain were necessary for collagen IV deposition and angiogenesis, whereas the SRCR domains were effective for these processes. Finally, surface coating with recombinant SRCR domains restored deposition of collagen IV by LOXL2-depleted cells. We thus propose that LOXL2 SRCR domains orchestrate scaffolding of the vascular basement membrane and angiogenesis through interactions with collagen IV and fibronectin, independently of the enzymatic cross-linking activity.

Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase.

PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2 Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.

Characterization of PlGoxB, a flavoprotein required for cysteine tryptophylquinone biosynthesis in glycine oxidase from Pseudoalteromonas luteoviolacea.

LodA-like proteins are oxidases with a protein-derived cysteine tryptophylquinone (CTQ) prosthetic group. In Pseudoalteromonas luteoviolacea glycine oxidase (PlGoxA), CTQ biosynthesis requires post-translational modifications catalyzed by a modifying enzyme encoded by PlgoxB. The PlGoxB protein was expressed and shown to possess a flavin cofactor. PlGoxB was unstable in solution as it readily lost the flavin and precipitated. PlGoxB precipitation was significantly reduced by incubation with either excess FAD or an equal concentration of prePlGoxA, the precursor protein that is its substrate. In contrast, the mature CTQ-bearing PlGoxA had no stabilizing effect. A homology model of PlGoxB was generated using the structure of Alkylhalidase CmIS. The FAD-binding site of PlGoxB in the model was nearly identical to that of the template structure. The bound FAD in PlGoxB had significant solvent exposure, consistent with the observed tendency to lose FAD. This also suggested that interaction of prePlGoxA with PlGoxB at the exposed FAD-binding site could prevent the observed loss of FAD and subsequent precipitation of PlGoxB. A docking model of the putative PlGoxB-prePlGoxA complex was consistent with these hypotheses. The experimental results and computational analysis implicate structural features of PlGoxB that contribute to its stability and function.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label.

1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

The Redox Properties of a Cysteine Tryptophylquinone-Dependent Glycine Oxidase Are Distinct from Those of Tryptophylquinone-Dependent Dehydrogenases.

GoxA is a cysteine tryptophylquinone (CTQ)-dependent glycine oxidase that is a member of a family of LodA-like proteins. The electrochemical midpoint potential ( Em) values for the quinone/semiquinone couple and the semiquinone/quinol couple were determined to be 111 and 21, respectively. The Em value for the overall two-electron quinone/quinol couple was similar to those of CTQ- and tryptophan tryptophylquinone (TTQ)-bearing dehydrogenases. However, for the well-studied TTQ-dependent methylamine dehydrogenase, the quinone/semiquinone couple is more negative than the semiquinone/quinol couple, the opposite of what was determined for GoxA. The change in Em value for the two-electron quinone/quinol couple of CTQ in GoxA with pH indicates that the overall two-electron transfer process is associated with the transfer of one proton. Thus, the quinol is anionic. The data reported herein further suggest that in GoxA the CTQ semiquinone is neutral, in contrast to the TTQ-dependent dehydrogenases, in which it is an anionic TTQ semiquinone. These results are discussed in the context of the structure and function of this glycine oxidase, compared to that of the tryptophylquinone-dependent dehydrogenases.

Structural and Spectroscopic Characterization of a Product Schiff Base Intermediate in the Reaction of the Quinoprotein Glycine Oxidase, GoxA.

The LodA-like proteins make up a recently identified family of enzymes that rely on a cysteine tryptophylquinone cofactor for catalysis. They differ from other tryptophylquinone enzymes in that they are oxidases rather than dehydrogenases. GoxA is a member of this family that catalyzes the oxidative deamination of glycine. Our previous work with GoxA from Pseudoalteromonas luteoviolacea demonstrated that this protein forms a stable intermediate upon anaerobic incubation with glycine. The spectroscopic properties of this species were unique among those identified for tryptophylquinone enzymes characterized to date. Here we use X-ray crystallography and resonance Raman spectroscopy to identify the GoxA catalytic intermediate as a product Schiff base. Structural work additionally highlights features of the active site pocket that confer substrate specificity, intermediate stabilization, and catalytic activity. The unusual properties of GoxA are discussed within the context of the other tryptophylquinone enzymes.

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

Extracellular Processing of Lysyl Oxidase-like 2 and Its Effect on Amine Oxidase Activity.

Overexpression of lysyl oxidase-like 2 (LOXL2) is associated with several hepatic and vascular fibrotic diseases and tumor progression in some aggressive cancers. Secreted LOXL2 promotes extracellular matrix cross-linking by catalyzing the oxidative deamination of peptidyl lysine. A great deal remains to be learned about the post-translational modifications of LOXL2, including whether such modifications modulate enzymatic and disease-promoting activities; such knowledge would inform the development of potential therapies. We discovered that upon secretion in cell culture, LOXL2 undergoes proteolytic processing of the first two of four scavenger receptor cysteine-rich domains at the N-terminus. A similar pattern of processing was also evident in tissue extracts from an invasive ductal carcinoma patient. Processing occurred at 314Arg-315Phe-316Arg-317Lys↓-318Ala-, implicating proprotein convertases. siRNA-mediated knockdown of proprotein convertases (furin, PACE4, and PC5/6), as well as incubation with their recombinant forms, showed that PACE4 is the major protease that acts on extracellular LOXL2. Unlike LOX, which requires cleavage of its propeptide for catalytic activation, cleavage of LOXL2 was not essential for tropoelastin oxidation or for cross-linking of collagen type IV in vitro. However, in the latter case, processing enhanced the extent of collagen cross-linking ∼2-fold at ≤10 nM LOXL2. These results demonstrate an important difference in the regulatory mechanisms for LOX and LOXL2 catalytic activity. Moreover, they pave the way for further studies of potential differential functions of LOXL2 isoforms in fibrosis and tumor progression.

Upregulation of MIAT Regulates LOXL2 Expression by Competitively Binding MiR-29c in Clear Cell Renal Cell Carcinoma.

BACKGROUND/AIMS: MIAT is a long noncoding RNA (lncRNA) involved in cell proliferation and the development of tumor. However, the exact effects and molecular mechanisms of MIAT in clear cell renal cell carcinoma (ccRCC) progression are still unknown. METHODS: We screened the lncRNAs' profile of ccRCC in The Cancer Genome Atlas database, and then examined the expression levels of lncRNA MIAT in 45 paired ccRCC tissue specimens and in cell lines by q-RT-PCR. MTS, colony formation, EdU, and Transwell assays were performed to examine the effect of MIAT on proliferation and metastasis of ccRCC. Western blot and luciferase assays were performed to determine whether MIAT can regulate Loxl2 expression by competitively binding miR-29c in ccRCC. RESULTS: MIAT was up-regulated in ccRCC tissues and cell lines. High MIAT expression correlated with worse clinicopathological features and shorter survival rate. Functional assays showed that knockdown of MIAT inhibited renal cancer cell proliferation and metastasis in vitro and in vivo. Luciferase and western blot assays further confirmed that miR-29c binds with MIAT. Additionally, the correlation of miR-29c with MIAT and Loxl2 was further verified in patients' samples. CONCLUSION: Our data indicated that MIAT might be an oncogenic lncRNA that promoted proliferation and metastasis of ccRCC, and could be a potential therapeutic target in human ccRCC.

Structure and Enzymatic Properties of an Unusual Cysteine Tryptophylquinone-Dependent Glycine Oxidase from Pseudoalteromonas luteoviolacea.

Glycine oxidase from Pseudoalteromonas luteoviolacea (PlGoxA) is a cysteine tryptophylquinone (CTQ)-dependent enzyme. Sequence analysis and phylogenetic analysis place it in a newly designated subgroup (group IID) of a recently identified family of LodA-like proteins, which are predicted to possess CTQ. The crystal structure of PlGoxA reveals that it is a homotetramer. It possesses an N-terminal domain with no close structural homologues in the Protein Data Bank. The active site is quite small because of intersubunit interactions, which may account for the observed cooperativy toward glycine. Steady-state kinetic analysis yielded the following values: kcat = 6.0 ± 0.2 s-1, K0.5 = 187 ± 18 µM, and h = 1.77 ± 0.27. In contrast to other quinoprotein amine dehydrogenases and oxidases that exhibit anomalously large primary kinetic isotope effects on the rate of reduction of the quinone cofactor by the amine substrate, no significant primary kinetic isotope effect was observed for this reaction of PlGoxA. The absorbance spectrum of glycine-reduced PlGoxA exhibits features in the range of 400-650 nm that have not previously been seen in other quinoproteins. Thus, in addition to the unusual structural features of PlGoxA, the kinetic and chemical reaction mechanisms of the reductive half-reaction of PlGoxA appear to be distinct from those of other amine dehydrogenases and amine oxidases that use tryptophylquinone and tyrosylquinone cofactors.

Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase.

Ethylene is an important phytohormone that promotes the ripening of fruits and senescence of flowers thereby reducing their shelf lives. Specific ethylene biosynthesis inhibitors would help to decrease postharvest loss. Here, we identify pyrazinamide (PZA), a clinical drug used to treat tuberculosis, as an inhibitor of ethylene biosynthesis in Arabidopsis thaliana, using a chemical genetics approach. PZA is converted to pyrazinecarboxylic acid (POA) in plant cells, suppressing the activity of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), the enzyme catalysing the final step of ethylene formation. The crystal structures of Arabidopsis ACO2 in complex with POA or 2-Picolinic Acid (2-PA), a POA-related compound, reveal that POA/2-PA bind at the active site of ACO, preventing the enzyme from interacting with its natural substrates. Our work suggests that PZA and its derivatives may be promising regulators of plant metabolism, in particular ethylene biosynthesis.