ABSTRACT
The IGF2 mRNA-binding protein 1 (IGF2BP1) is a non-catalytic post-transcriptional enhancer of tumor growth upregulated and associated with adverse prognosis in solid cancers. However, conserved effector pathway(s) and the feasibility of targeting IGF2BP1 in cancer remained elusive. We reveal that IGF2BP1 is a post-transcriptional enhancer of the E2F-driven hallmark in solid cancers. IGF2BP1 promotes G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators of this checkpoint like E2F1. This IGF2BP1-driven shortening of the G1 cell cycle phase relies on 3'UTR-, miRNA- and m6A-dependent regulation and suggests enhancement of cell cycle progression by m6A-modifications across cancers. In addition to E2F transcription factors, IGF2BP1 also stabilizes E2F-driven transcripts directly indicating post-transcriptional 'super'-enhancer role of the protein in E2F-driven gene expression in cancer. The small molecule BTYNB disrupts this enhancer function by impairing IGF2BP1-RNA association. Consistently, BTYNB interferes with E2F-driven gene expression and tumor growth in experimental mouse tumor models.
Subject(s)
E2F Transcription Factors/genetics , Neoplasms/genetics , RNA-Binding Proteins/genetics , 3' Untranslated Regions/genetics , Animals , Cell Line, Tumor , E2F1 Transcription Factor/genetics , Gene Expression Regulation, Neoplastic/genetics , Humans , Mice , Neoplasms/pathology , RNA-Binding Proteins/drug effects , Small Molecule Libraries/pharmacologyABSTRACT
Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum. Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic organic compounds, including (E)-chalcones, (E)-N'-benzylidene-benzohydrazides and alkyl-esters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC50 values (8.5 ± 0.8 µM, 9.5 ± 0.2 µM and 4.9 ± 1.3 µM, respectively). The best inhibitor (compound 66) demonstrated non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy assays, we suggest a potential allosteric site for the interaction of this compound, located between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural biology tools and mass spectrometry to characterize the inhibition mechanisms of novel compounds targeting FP-2.
Subject(s)
Antimalarials/chemical synthesis , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Drug Discovery/methods , Plasmodium falciparum/drug effects , Small Molecule Libraries/chemistry , Antimalarials/chemistry , Antimalarials/pharmacology , Cysteine Endopeptidases/genetics , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Escherichia coli/genetics , Molecular Structure , Plasmodium falciparum/enzymology , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Lipases are well-known enzymes extensively used in industrial biotransformation processes. Besides, their structural and catalytic characteristics have attracted increasing attention of several industries in the last years. In this work, we used biophysical and molecular modeling tools to assess structural properties of Staphylococcus xylosus lipase (SXL). We studied the thermal unfolding of this protein and its zinc-dependent thermotolerance. We demonstrated that SXL is able to be active and stable at moderate temperatures, but this feature is only acquired in the presence of Zn(2+). Such characteristic indicates SXL as a zinc-dependent metallolipase.
Subject(s)
Lipase/chemistry , Staphylococcus/enzymology , Zinc/chemistry , Amino Acid Sequence , Enzyme Stability , Lipolysis , Models, Molecular , Molecular Sequence DataABSTRACT
Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42 degrees C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80 degrees C all enzymes retained their initial activities.
Subject(s)
Bacterial Proteins/chemistry , Lipase/chemistry , Lipase/genetics , Recombinant Proteins/chemistry , Staphylococcus/enzymology , Amino Acid Substitution , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Enzyme Stability , Escherichia coli , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Mutation , Point Mutation , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , TemperatureABSTRACT
Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.
Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Lipase/isolation & purification , Lipase/metabolism , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Staphylococcus/enzymology , Bacterial Proteins/genetics , Chromatography, Gel , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Lipase/genetics , Recombinant Fusion Proteins/genetics , Staphylococcus/genetics , Substrate Specificity , TemperatureABSTRACT
Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His(6)-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40 degrees C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.
Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Esterases/isolation & purification , Esterases/metabolism , Lactobacillus plantarum/enzymology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Butyrates/metabolism , Cloning, Molecular , DNA, Bacterial , Escherichia coli/genetics , Esterases/chemistry , Esterases/genetics , Genes, Bacterial , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Lactobacillus plantarum/genetics , Molecular Sequence Data , Molecular Weight , Nitrophenols/metabolism , Plasmids , Protein Structure, Secondary/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Spectrophotometry, Ultraviolet , Substrate Specificity , TemperatureABSTRACT
Transthyretin-related proteins (TRPs) constitute a family of proteins structurally related to transthyretin (TTR) and are found in a large range of bacterial, fungal, plant, invertebrate, and vertebrate species. However, it was recently recognized that both prokaryotic and eukaryotic members of this family are not functionally related to transthyretins. TRPs are in fact involved in the purine catabolic pathway and function as hydroxyisourate hydrolases. An open reading frame encoding a protein similar to the Escherichia coli TRP was identified in Herbaspirillum seropedicae genome (Hs_TRP). It was cloned, overexpressed in E. coli, and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein, and circular dichroism spectrum indicated a predominance of beta-sheet structure, as expected for a TRP. We have demonstrated that Hs_TRP is a 5-hydroxyisourate hydrolase and by site-directed mutagenesis the importance of three conserved catalytic residues for Hs_TRP activity was further confirmed. The production of large quantities of this recombinant protein opens up the possibility of obtaining its 3D-structure and will help further investigations into purine catabolism.
Subject(s)
Herbaspirillum/metabolism , Prealbumin/biosynthesis , Amino Acid Sequence , Circular Dichroism , Molecular Sequence Data , Mutation , Open Reading Frames , Prealbumin/chemistry , Prealbumin/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Landfill leachates are liquid effluents with elevated concentrations of chemical compounds that can cause serious environmental pollution. In the south of the state of Santa Catarina, Brazil, a sanitary landfill was installed that employs a system of anaerobic/facultative lagoons for the treatment of its leachate. The present work examined the toxic and genotoxic potential of untreated and treated landfill leachates using bioassays. The chemical, toxic, genotoxic and mutagenic properties of the untreated leachate and the treated leachate were determined. Examination of the chemical properties showed a marked decrease in parameters after treatment, as well as in toxicity towards all the organisms tested. The results of the comet assay demonstrated that both leachates showed genotoxicity in all of the organisms tested, indicating the persistence of genotoxic substances even after treatment. A significant decrease in micronucleated cells was detected in Geophagus brasiliensis exposed to the treated leachate compared to untreated.
