Distinct Enzymes Research Articles

NADPH oxidases and ROS signaling in the gastrointestinal tract.

Reactive oxygen species (ROS), initially categorized as toxic by-products of aerobic metabolism, have often been called a double-edged sword. ROS are considered indispensable when host defense and redox signaling is concerned and a threat in inflammatory or degenerative diseases. This generalization does not take in account the diversity of oxygen metabolites being generated, their physicochemical characteristics and their production by distinct enzymes in space and time. NOX/DUOX NADPH oxidases are the only enzymes solely dedicated to ROS production and the prime ROS producer for intracellular and intercellular communication due to their widespread expression and intricate regulation. Here we discuss new insights of how NADPH oxidases act via ROS as multifaceted regulators of the intestinal barrier in homeostasis, infectious disease and intestinal inflammation. A closer look at monogenic VEOIBD and commensals as ROS source supports the view of H2O2 as key beneficial messenger in the barrier ecosystem.

Mechanistic comparison of human DNA Ligases I and III

DNA ligation is an essential step to complete DNA repair, replication and recombination processes. The human genome encodes two divergent, yet structurally similar DNA ligases, LIG1 and LIG3. Each enzyme has unique amino and carboxy‐terminal extensions and the conserved catalytic core is only 22% identical. LIG3 is essential for mitochondrial genome maintenance, but is also expressed ubiquitously as a nuclear isoform. To understand the mechanism and fidelity of human DNA ligation we are working to characterize DNA LIG1 and LIG3 and their associated protein complexes. Transient and steady state kinetic approaches are used to elucidate distinct enzyme conformational changes and the role of the essential metal ions in substrate selection and catalysis. By determining the similarities and differences between these DNA ligase isoforms, we provide new insights into the clinical situations in which mutation or changes in gene expression are associated with diseases such as cancer and primary immune deficiency.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

α-cyano-4-hydroxycinnamate impairs pancreatic cancer cells by stimulating the p38 signaling pathway

Multiple studies are currently targeting dysregulated cancer cell metabolism with distinct combinations of inhibitors. In this study, we evaluated in pancreatic cancer cells metformin, which blocks oxidative phosphorylation, in combination with α-cyano-4-hydroxycinnamate, which has been reported to inhibit the export of lactate from the cytosol. The combination of metformin with α-cyano-4-hydroxycinnamate had a major inhibitory effect on the migration of 6606PDA cells. Monotherapy with α-cyano-4-hydroxycinnamate and especially the combination with metformin also caused significant inhibition of cell proliferation and induced cell death. α-cyano-4-hydroxycinnamate in combination with metformin reduced the export of lactate significantly, whereas α-cyano-4-hydroxycinnamate monotherapy only modestly influenced lactate export. None of these two drugs inhibited the expression of distinct glycolytic enzymes. Interestingly, α-cyano-4-hydroxycinnamate rather inhibited the ERK and very strongly stimulated the p38 signaling pathway in 6606PDA as well as in 7265PDA cells. In addition, the inhibition of the p38 signaling pathway by PH-797804 partially reversed the effect of α-cyano-4-hydroxycinnamate on cell apoptosis in both cell lines. We conclude that α-cyano-4-hydroxycinnamate monotherapy and especially the combinatorial therapy with metformin has strong anti-cancerous effects. α-cyano-4-hydroxycinnamate causes cancer cell apoptosis by a novel mechanism for this drug, namely the stimulation of the p38 signaling pathway.

Dynamical origins of heat capacity changes in enzyme-catalysed reactions

Heat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed, along with changes in energetic fluctuations across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.

Substitution of Alanine at Position 184 with Glutamic Acid in Escherichia coli PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity.

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic β-lactam resistance. Though PBP5 does not have β-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A β-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in β-lactamases. It is speculated that β-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ∆PBP5 mutant elevates the β-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and β-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.

Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

Accumulating data support a role for bioactive lipids as mediators of lipotixicity in cardiomyocytes. One class of these, the ceramides, constitutes a family of molecules that differ in structure and are synthesized by distinct enzymes, ceramide synthase (CerS)1-CerS6. Data support that specific ceramides and the enzymes that catalyze their formation play distinct roles in cell function. In a mouse model of diabetic cardiomyopathy, sphingolipid profiling revealed increases in not only the CerS5-derived ceramides but also in very long chain (VLC) ceramides derived from CerS2. Overexpression of CerS2 elevated VLC ceramides caused insulin resistance, oxidative stress, mitochondrial dysfunction, and mitophagy. Palmitate induced CerS2 and oxidative stress, mitophagy, and apoptosis, which were prevented by depletion of CerS2. Neither overexpression nor knockdown of CerS5 had any function in these processes, suggesting a chain-length dependent impact of ceramides on mitochondrial function. This concept was also supported by the observation that synthetic mitochondria-targeted ceramides led to mitophagy in a manner proportional to N-acyl chain length. Finally, blocking mitophagy exacerbated cell death. Taken together, our results support a model by which CerS2 and VLC ceramides have a distinct role in lipotoxicity, leading to mitochondrial damage, which results in subsequent adaptive mitophagy. Our data reveal a novel lipotoxic pathway through CerS2.-Law, B. A., Liao, X., Moore, K. S., Southard, A., Roddy, P., Ji, R., Szulc, Z., Bielawska, A., Schulze, P. C., Cowart, L. A. Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates During E3-Mediated Ubiquitin-Like Protein Ligation.

Most cellular functions rely on pathways that catalyze posttranslational modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. Like other posttranslational modifications that require distinct writers, readers, and erasers during signaling, Ub/Ubl pathways employ distinct enzymes that catalyze Ub/Ubl attachment, Ub/Ubl recognition, and Ub/Ubl removal. Ubl protein conjugation typically relies on parallel but distinct enzymatic cascades catalyzed by an E1-activating enzyme, an E2 carrier protein, and an E3 ubiquitin-like protein ligase. One major class of E3, with ca. 600 members, harbors RING or the RING-like SP-RING or Ubox domains. These RING/RING-like domains bind and activate the E2-Ubl thioester by stabilizing a conformation that is optimal for nucleophilic attack by the side chain residue (typically lysine) on the substrate. These RING/RING-like domains typically function together with other domains or protein complexes that often serve to recruit particular substrates. How these RING/RING-like E3 domains function to activate the E2-Ubl thioester while engaged with substrate remains poorly understood. We describe a strategy to generate and purify a unique E2Ubc9-UblSUMO thioester mimetic that can be cross-linked to the SubstratePCNA at Lys164, a conjugation site that is only observed in the presence of E3Siz1. We describe two techniques to cross-link the E2Ubc9-UblSUMO thioester mimetic active site to the site of modification on PCNA and the subsequent purification of these complexes. Finally, we describe the reconstitution and purification of the E2Ubc9-UblSUMO-PCNA complex with the E3Siz1 and purification that enabled its crystallization and structure determination. We think this technique can be extended to other E2-Ubl-substrate/E3 complexes to better probe the function and specificity of RING-based E3 Ubl ligases.

Functional Involvement of Leishmania donovani Tryparedoxin Peroxidases during Infection and Drug Treatment.

The parasite Leishmania donovani causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in Leishmania showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

ABSTRACTAminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.

Comparative evaluation of Populus variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems

BackgroundPopulus natural variants have been shown to realize a broad range of sugar yields during saccharification, however, the structural features responsible for higher sugar release from natural variants are not clear. In addition, the sugar release patterns resulting from digestion with two distinct biological systems, fungal enzymes and Clostridium thermocellum, have yet to be evaluated and compared. This study evaluates the effect of structural features of three natural variant Populus lines, which includes the line BESC standard, with respect to the overall process of sugar release for two different biological systems.ResultsPopulus natural variants, SKWE 24-2 and BESC 876, showed higher sugar release from hydrothermal pretreatment combined with either enzymatic hydrolysis or Clostridium thermocellum fermentation compared to the Populus natural variant, BESC standard. However, C. thermocellum outperformed the fungal cellulases yielding 96.0, 95.5, and 85.9% glucan plus xylan release from SKWE 24-2, BESC 876, and BESC standard, respectively. Among the feedstock properties evaluated, cellulose accessibility and glycome profiling provided insights into factors that govern differences in sugar release between the low recalcitrant lines and the BESC standard line. However, because this distinction was more apparent in the solids after pretreatment than in the untreated biomass, pretreatment was necessary to differentiate recalcitrance among Populus lines. Glycome profiling analysis showed that SKWE 24-2 contained the most loosely bound cell wall glycans, followed by BESC 876, and BESC standard. Additionally, lower molecular weight lignin may be favorable for effective hydrolysis, since C. thermocellum reduced lignin molecular weight more than fungal enzymes across all Populus lines.ConclusionsLow recalcitrant Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar yields than BESC standard when hydrothermal pretreatment was combined with biological digestion. However, C. thermocellum was determined to be a more robust and effective biological catalyst than a commercial fungal cellulase cocktail. As anticipated, recalcitrance was not readily predicted through analytical methods that determined structural properties alone. However, combining structural analysis with pretreatment enabled the identification of attributes that govern recalcitrance, namely cellulose accessibility, xylan content in the pretreated solids, and non-cellulosic glycan extractability.

Cross-species complementation of bacterial- and eukaryotic-type cardiolipin synthases.

The glycerophospholipid cardiolipin is a unique constituent of bacterial and mitochondrial membranes. It is involved in forming and stabilizing high molecular mass membrane protein complexes and in maintaining membrane architecture. Absence of cardiolipin leads to reduced efficiency of the electron transport chain, decreased membrane potential, and, ultimately, impaired respiratory metabolism. For the protozoan parasite Trypanosoma brucei cardiolipin synthesis is essential for survival, indicating that the enzymes involved in cardiolipin production represent potential drug targets. T. brucei cardiolipin synthase (TbCLS) is unique as it belongs to the family of phospholipases D (PLD), harboring a prokaryotic-type cardiolipin synthase (CLS) active site domain. In contrast, most other eukaryotic CLS, including the yeast ortholog ScCrd1, are members of the CDP-alcohol phosphatidyltransferase family. To study if these mechanistically distinct CLS enzymes are able to catalyze cardiolipin production in a cell that normally expresses a different type of CLS, we expressed TbCLS and ScCrd1 in CLS-deficient yeast and trypanosome strains, respectively. Our results show that TbCLS complemented cardiolipin production in CRD1 knockout yeast and partly restored wild-type colony forming capability under stress conditions. Remarkably, CL remodeling appeared to be impaired in the transgenic construct, suggesting that CL production and remodeling are tightly coupled processes that may require a clustering of the involved proteins into specific CL-synthesizing domains. In contrast, no complementation was observed by heterologous expression of ScCrd1 in conditional TbCLS knockout trypanosomes, despite proper mitochondrial targeting of the protein.

The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid

The periplasmic conversion of ferribactin to pyoverdine is essential for siderophore biogenesis in fluorescent pseudomonads, such as pathogenic Pseudomonas aeruginosa or plant growth-promoting Pseudomonas fluorescens The non-ribosomal peptide ferribactin undergoes cyclizations and oxidations that result in the fluorophore, and a strictly conserved fluorophore-bound glutamic acid residue is converted to a range of variants, including succinamide, succinic acid, and α-ketoglutaric acid residues. We recently discovered that the pyridoxal phosphate-containing enzyme PvdN is responsible for the generation of the succinamide, which can be hydrolyzed to succinic acid. Based on this, a distinct unknown enzyme was postulated to be responsible for the conversion of the glutamic acid to α-ketoglutaric acid. Here we report the identification and characterization of this enzyme in P. fluorescens strain A506. In silico analyses indicated a periplasmic transaminase in fluorescent pseudomonads and other proteobacteria that we termed PtaA for "periplasmic transaminase A" An in-frame-deleted ptaA mutant selectively lacked the α-ketoglutaric acid form of pyoverdine, and recombinant PtaA complemented this phenotype. The ptaA/pvdN double mutant produced exclusively the glutamic acid form of pyoverdine. PtaA is homodimeric and contains a pyridoxal phosphate cofactor. Mutation of the active-site lysine abolished PtaA activity and affected folding as well as Tat-dependent transport of the enzyme. In pseudomonads, the occurrence of ptaA correlates with the occurrence of α-ketoglutaric acid forms of pyoverdines. As this enzyme is not restricted to pyoverdine-producing bacteria, its catalysis of periplasmic transaminations is most likely a general tool for specific biosynthetic pathways.

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model.

Oxidative stress including iron excess has been associated with carcinogenesis. The level of 8-oxoguanine, a major oxidatively modified base in DNA, is maintained very low by three distinct enzymes, encoded by OGG1, MUTYH and MTH1. Germline biallelic inactivation of MUTYH represents a familial cancer syndrome called MUTYH-associated polyposis. Here, we used Mutyh-deficient mice to evaluate renal carcinogenesis induced by ferric nitrilotriacetate (Fe-NTA). Although the C57BL/6 background is cancer-resistant, a repeated intraperitoneal administration of Fe-NTA induced a high incidence of renal cell carcinoma (RCC; 26.7%) in Mutyh-deficient mice in comparison to wild-type mice (7.1%). Fe-NTA treatment also induced renal malignant lymphoma, which did not occur without the Fe-NTA treatment in both the genotypes. Renal tumor-free survival after Fe-NTA treatment was marginally different (P = 0.157) between the two genotypes. Array-based comparative genome hybridization analyses revealed, in RCC, the loss of heterozygosity in chromosomes 4 and 12 without p16INKA inactivation; these results were confirmed by a methylation analysis and showed no significant difference between the genotypes. Lymphomas showed a preference for genomic amplifications. Dlk1 inactivation by promoter methylation may be involved in carcinogenesis in both tumors. Fe-NTA-induced murine RCCs revealed significantly less genomic aberrations than those in rats, demonstrating a marked species difference.

Structural and Functional Characterization of a Short-Chain Flavodoxin Associated with a Noncanonical 1,2-Propanediol Utilization Bacterial Microcompartment.

Bacterial microcompartments (BMCs) are proteinaceous organelles that encapsulate enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Metabolosomes share a common core of enzymes and a distinct signature enzyme for substrate degradation that defines the function of the BMC (e.g., propanediol or ethanolamine utilization BMCs, or glycyl-radical enzyme microcompartments). Loci encoding metabolosomes also typically contain genes for proteins that support organelle function, such as regulation, transport of substrate, and cofactor (e.g., vitamin B12) synthesis and recycling. Flavoproteins are frequently among these ancillary gene products, suggesting that these redox active proteins play an undetermined function in many metabolosomes. Here, we report the first characterization of a BMC-associated flavodoxin (Fld1C), a small flavoprotein, derived from the noncanonical 1,2-propanediol utilization BMC locus (PDU1C) of Lactobacillus reuteri. The 2.0 Å X-ray structure of Fld1C displays the α/β flavodoxin fold, which noncovalently binds a single flavin mononucleotide molecule. Fld1C is a short-chain flavodoxin with redox potentials of -240 ± 3 mV oxidized/semiquinone and -344 ± 1 mV semiquinone/hydroquinone versus the standard hydrogen electrode at pH 7.5. It can participate in an electron transfer reaction with a photoreductant to form a stable semiquinone species. Collectively, our structural and functional results suggest that PDU1C BMCs encapsulate Fld1C to store and transfer electrons for the reactivation and/or recycling of the B12 cofactor utilized by the signature enzyme.

Indoleamine 2,3-Dioxygenase and Its Therapeutic Inhibition in Cancer.

The tryptophan catabolic enzyme indoleamine 2,3-dioxygenase-1 (IDO1) has attracted enormous attention in driving cancer immunosuppression, neovascularization, and metastasis. IDO1 suppresses local CD8+ T effector cells and natural killer cells and induces CD4+ T regulatory cells (iTreg) and myeloid-derived suppressor cells (MDSC). The structurally distinct enzyme tryptophan dioxygenase (TDO) also has been implicated recently in immune escape and metastatic progression. Lastly, emerging evidence suggests that the IDO1-related enzyme IDO2 may support IDO1-mediated iTreg and contribute to B-cell inflammed states in certain cancers. IDO1 and TDO are upregulated widely in neoplastic cells but also variably in stromal, endothelial, and innate immune cells of the tumor microenviroment and in tumor-draining lymph nodes. Pharmacological and genetic proofs in preclinical models of cancer have validated IDO1 as a cancer therapeutic target. IDO1 inhibitors have limited activity on their own but greatly enhance "immunogenic" chemotherapy or immune checkpoint drugs. IDO/TDO function is rooted in inflammatory programming, thereby influencing tumor neovascularization, MDSC generation, and metastasis beyond effects on adaptive immune tolerance. Discovery and development of two small molecule enzyme inhibitors of IDO1 have advanced furthest to date in Phase II/III human trials (epacadostat and navoximod, respectively). Indoximod, a tryptophan mimetic compound with a different mechanism of action in the IDO pathway has also advanced in multiple Phase II trials. Second generation combined IDO/TDO inhibitors may broaden impact in cancer treatment, for example, in addressing IDO1 bypass (inherent resistance) or acquired resistance to IDO1 inhibitors. This review surveys knowledge about IDO1 function and how IDO1 inhibitors reprogram inflammation to heighten therapeutic responses in cancer.

Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei: Deviation from the Norm.

ABSTRACTThe trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium.

Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m3C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m3C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m3C formation in mammals as well. Here, we report the discovery and characterization of three distinct m3C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m3C in specific tRNAs and that METTL8 only contributes m3C to mRNA. MS analysis revealed that there is an ∼30–40% and ∼10–15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m3C in total tRNA, and primer extension analysis located METTL2-modified m3C at position 32 of tRNAThr isoacceptors and tRNAArg(CCU). We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m3C modification in mRNA, and the discovery of METTL8 as an mRNA m3C writer enzyme opens the door to future studies of other m3C epitranscriptomic reader and eraser functions.

Chromatin remodeling: a complex affair.

ATP‐dependent chromatin remodelers are multi‐subunit enzymes that catalyze nucleosome dynamics essential for chromosomal functions, and their inactivation or dysregulation can lead to numerous diseases, including neuro‐degenerative disorders and cancers. Each remodeler contains a conserved ATPase “motor” whose activity or targeting can be regulated by enzyme‐specific, accessory subunits. The human ISWI subfamily of remodelers has been defined as a group of more than six different enzyme complexes where one of two related ATPase subunits (Snf2L/SMARCA1 and Snf2H/SMARCA5) is paired with one of six different accessory subunits. In this issue of EMBO Reports , Oppikofer et al [1] find that the human ISWI subfamily is even more polymorphic in nature—every known accessory subunit can interact and function with both ATPase isoforms. This raises the complexity of the human ISWI subfamily to > 12 distinct enzymes, with the possibility for much higher levels of combinatorial assemblies, and has the potential to create enzymes with novel biochemical activities, as well as novel regulatory wiring through differential interactions with locus‐specific factors or histone modifications.

Profiling of metalloprotease activities in cerebrospinal fluids of patients with neoplastic meningitis

BackgroundNeoplastic invasion into leptomeninges and subarachnoid space, resulting in neoplastic meningitis (NM) is a fatal complication of advanced solid and hematological neoplasms. Identification of malignant involvement of the cerebrospinal fluid (CSF) early in the disease course has crucial prognostic and therapeutic implications, but remains challenging. As indicators of extracellular matrix (ECM) degradation and breakdown of the blood–brain-barrier, Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloproteases (ADAMs) are potential analytes for cerebral pathophysiology and metastatic dissemination of tumor cells into the CSF.MethodsWe compared protease activities in CSF samples from patients with NM and control individuals using FRET-based metalloprotease substrates with distinct enzyme selectivity profiles in a real-time, multiplex approach termed “proteolytic activity matrix assay” (PrAMA). Protease activity dynamics can be tracked by fluorescence changes over time. By simultaneously monitoring a panel of 5 FRET-substrate cleavages, a proteolytic signature can be identified and analyzed to infer the activities of multiple specific proteases. Distinct patterns of substrate cleavage comparing disease vs. control samples allow rapid, reproducible and sensitive discrimination even in small volumes of CSF.ResultsIndividual substrate cleavage rates were linked to distinct proteases, and PrAMA computational inference implied increased activities of MMP-9, ADAM8 and ADAM17 (4–5-fold on average) in CSF samples from NM patients that were inhibitable by the metalloprotease inhibitor batimastat (BB-94). The activities of these proteases correlated with blood–brain barrier impairment. Notably, CSF cell counts were not found to directly reflect the protease activities observed in CSF samples from NM patients; this may explain the frequent clinical observation of negative cytology in NM patients.ConclusionPrAMA analysis of CSF samples is a potential diagnostic method for sensitive detection of NM and may be suitable for the clinical routine.

1,2,4-Triazole-3-thione Compounds as Inhibitors of Dizinc Metallo-β-lactamases.

Metallo-β-lactamases (MBLs) cause resistance of Gram-negative bacteria to β-lactam antibiotics and are of serious concern, because they can inactivate the last-resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4-amino-2,4-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non-amino compound IIIB (1,2-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3 D structures suggested that the triazole-thione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC50 values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM-4, VIM-2, NDM-1, and IMP-1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM-1, VIM-2, and IMP-1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc-dependent hydrolases belonging to the MBL-fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC50 values toward plasmodial glyoxalase II were in the 10 μm range.