RESUMEN
Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases.
RESUMEN
Peptidoglycan hydrolytic enzymes are considered to be a promising alternative to conventional antibiotics in combating bacterial infections. To identify novel hydrolytic enzymes, we performed a database search with the sequences of two thermostable endolysins with high bactericidal activity, studied earlier in our laboratory. Both these enzymes originate from Thermus scotoductus bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from Clostridium intestinale URNW was found to have the highest amino acid sequence similarity to the bacteriophage proteins and was chosen for further analysis. The recombinant enzyme showed strong activity against its host bacteria C. intestinale, as well as against C. sporogenes, Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus, on average causing a 5.12 ± 0.14 log reduction of viable S. aureus ATCC 25923 cells in a bactericidal assay. Crystallographic studies of the protein showed that the catalytic site of LysC contained a zinc atom coordinated by amino acid residues His50, His147, and Cys155, a feature characteristic for type 2 amidases. Surprisingly, neither of these residues, nor any other of the four conserved residues in the vicinity of the active site, His51, Thr52, Tyr76, and Thr153, were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged N-terminal region of the enzyme. Potential antibacterial activity of this part of the sequence, predicted by the Antimicrobial Sequence Scanning System, AMPA, was confirmed in our experimental studies; the truncated version of LysC (LysCΔ2-23) completely lacked antibacterial activity. Moreover, a synthetic peptide, which we termed Intestinalin, with a sequence identical to the first thirty amino acids of LysC, displayed substantial anti-staphylococcal activity with IC50 of 6 µg/mL (1.5 µM). This peptide was shown to have α-helical conformation in solution in the presence of detergents which is a common feature of amphipathic α-helical antimicrobial peptides.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Clostridium/enzimología , Endopeptidasas/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Bacteriófagos/enzimología , Dominio Catalítico , Simulación por Computador , Secuencia Conservada , Cristalografía por Rayos X , Endopeptidasas/química , Endopeptidasas/genética , Endopeptidasas/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacología , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Staphylococcus aureus/efectos de los fármacos , Proteínas Virales/químicaRESUMEN
Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN42-210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with â¼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN42-210]24·[NFS1]24·[ISD11]24·[ISCU]24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN42-210]24·[ISCU]24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42-210 trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2 sub-complexes to the surface results in a globular macromolecule with a diameter of â¼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN42-210 to ISCU.
Asunto(s)
Liasas de Carbono-Azufre/química , Proteínas de Unión a Hierro/química , Proteínas Reguladoras del Hierro/química , Proteínas Hierro-Azufre/química , Proteínas Mitocondriales/química , Simulación de Dinámica Molecular , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Proteínas Reguladoras del Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , FrataxinaRESUMEN
The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of â¼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly.
Asunto(s)
Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Azufre/metabolismo , Cristalografía por Rayos X , Proteínas de Unión a Hierro/química , Proteínas Hierro-Azufre/química , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , FrataxinaRESUMEN
Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.
Asunto(s)
Ferroquelatasa/química , Ferroquelatasa/metabolismo , Hemo/biosíntesis , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/crecimiento & desarrollo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , FrataxinaRESUMEN
The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.
Asunto(s)
Inhibidores Enzimáticos/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/química , Espermidina Sintasa/química , Desoxiadenosinas/química , Estructura Terciaria de Proteína , Putrescina/química , Espermidina/química , Espermidina Sintasa/antagonistas & inhibidores , Tionucleósidos/químicaRESUMEN
The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Unión a Hierro/química , Hierro/química , Multimerización de Proteína , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Proteínas de Unión a Hierro/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Termodinámica , FrataxinaRESUMEN
The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg(2+) into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of â¼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of â¼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.
Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/enzimología , Liasas/química , Modelos Moleculares , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5â Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.
Asunto(s)
Bacteriófagos , ADN Cruciforme , Archaea/genética , Archaea/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Resolvasas de Unión Holliday/química , Resolvasas de Unión Holliday/genética , Resolvasas de Unión Holliday/metabolismo , Thermus thermophilusRESUMEN
This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19â Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97â Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.
Asunto(s)
Bacteriófagos , ADN Polimerasa I , ADN Polimerasa I/química , ADN Polimerasa I/genética , Fosfodiesterasa I , Thermus , Polimerasa Taq/química , Escherichia coliRESUMEN
In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.
Asunto(s)
Leucemia Mieloide Aguda , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Animales , Humanos , Ratones , Antivirales/farmacología , Dihidroorotato Deshidrogenasa , Dipiridamol/uso terapéutico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Piridinas/farmacología , Piridinas/uso terapéutico , Relación Estructura-ActividadRESUMEN
Ferrochelatase catalyzes the insertion of Fe(2+) into protoporphyrin IX. The enzymatic product heme (protoheme IX) is a well-known cofactor in a wide range of proteins. The insertion of metal ions other than Fe(2+) occurs rarely in vivo, but all ferrochelatases that have been studied can insert Zn(2+) at a good rate in vitro. Co(2+), but not Cu(2+), is known to be a good substrate of the mammalian and Saccharomyces cerevisiae ferrochelatases. In contrast, Cu(2+), but not Co(2+), has been found to be a good substrate of bacterial Bacillus subtilis ferrochelatase. It is not known how ferrochelatase discriminates between different metal ion substrates. Structural analysis of B. subtilis ferrochelatase has shown that Tyr13 is an indirect ligand of Fe(2+) and a direct ligand of a copper mesoporphyrin product. A structure-based comparison revealed that Tyr13 aligns with a Met residue in the S. cerevisiae and human ferrochelatases. Tyr13 was changed to Met in the B. subtilis enzyme by site-directed mutagenesis. Enzymatic measurements showed that the modified enzyme inserted Co(2+) at a higher rate than the wild-type B. subtilis ferrochelatase, but it had lost the ability to use Cu(2+) as a substrate. Thus, the B. subtilis Tyr13Met ferrochelatase showed the same metal specificity as that of the ferrochelatases from S. cerevisiae and human.
Asunto(s)
Bacillus subtilis/enzimología , Ferroquelatasa/química , Ferroquelatasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cobalto/metabolismo , Cobre/metabolismo , Ferroquelatasa/genética , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Especificidad por Sustrato , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMEN
Chelatases catalyze the insertion of a specific metal ion into porphyrins, a key step in the synthesis of metalated tetrapyrroles that are essential for many cellular processes. Despite apparent common structural features among chelatases, no general reaction mechanism accounting for metal ion specificity has been established. We propose that chelatase-induced distortion of the porphyrin substrate not only enhances the reaction rate by decreasing the activation energy of the reaction but also modulates which divalent metal ion is incorporated into the porphyrin ring. We evaluate the recently recognized interaction between ferrochelatase and frataxin as a way to regulate iron delivery to ferrochelatase, and thus iron and heme metabolism. We postulate that the ferrochelatase-frataxin interaction controls the type of metal ion that is delivered to ferrochelatase.
Asunto(s)
Ferroquelatasa/metabolismo , Animales , Catálisis , Ferroquelatasa/química , Humanos , Proteínas de Unión a Hierro/metabolismo , Metales/química , Metales/metabolismo , Porfirinas/metabolismo , Unión Proteica , FrataxinaRESUMEN
The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1, a potent hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 µM).
Asunto(s)
Compuestos de Bifenilo/química , Inhibidores Enzimáticos/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Pirazoles/química , Piridinas/química , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Dihidroorotato Deshidrogenasa , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Semivida , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pirazoles/metabolismo , Pirazoles/farmacología , Pirazoles/uso terapéutico , Piridinas/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
Asunto(s)
Genoma Viral/genética , Metagenómica , Bioprospección/organización & administración , Biología Computacional , Bases de Datos Genéticas , Europa (Continente) , Respiraderos Hidrotermales/virología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Viroma/genética , Virus/clasificación , Virus/genéticaRESUMEN
Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.
Asunto(s)
Proteínas Bacterianas/química , Brucella melitensis/enzimología , Liasas/química , Chaperonas Moleculares/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Clonación Molecular , Liasas/genética , Microscopía Electrónica/métodos , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPß were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3â Å, respectively. XepA consists of two antiparallel ß-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9â kDa per monomer), which is less than half the size of XepA (30.3â kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each ß-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.
Asunto(s)
Fagos de Bacillus/metabolismo , Profagos/metabolismo , Proteínas Virales/química , Bacillus subtilis/virología , Clonación Molecular , Cristalografía por Rayos X/métodos , Estructura Terciaria de ProteínaRESUMEN
Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) has been clinically validated as a target for antimalarial drug discovery, as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. Here, we have identified new hydroxyazole scaffold-based PfDHODH inhibitors belonging to two different chemical series. The first series was designed by a scaffold hopping strategy that exploits the use of hydroxylated azoles. Within this series, the hydroxythiadiazole 3 was identified as the best selective PfDHODH inhibitor (IC50 12.0⯵M). The second series was designed by modulating four different positions of the hydroxypyrazole scaffold. In particular, hydroxypyrazoles 7e and 7f were shown to be active in the low µM range (IC50 2.8 and 5.3⯵M, respectively). All three compounds, 3, 7e and 7f showed clear selectivity over human DHODH (IC50â¯>â¯200⯵M), low cytotoxicity, and retained micromolar activity in P. falciparum-infected erythrocytes. The crystallographic structures of PfDHODH in complex with compounds 3 and 7e proved their binding mode, supplying essential data for future optimization of these scaffolds.
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Antimaláricos/química , Inhibidores Enzimáticos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Antimaláricos/farmacología , Azoles/química , Azoles/farmacología , Sitios de Unión , Cristalografía por Rayos X , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Eritrocitos/parasitología , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Unión Proteica , Pirazoles/química , Pirazoles/farmacología , Relación Estructura-ActividadRESUMEN
Inhibitors of dihydroorotate dehydrogenase (DHODH) have been suggested for the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections. Here we present the structures of N-terminally truncated (residues Met30-Arg396) DHODH in complex with two inhibitors: a brequinar analogue (6) and a novel inhibitor (a fenamic acid derivative) (7), as well as the first structure of the enzyme to be characterized without any bound inhibitor. It is shown that 7 uses the "standard" brequinar binding mode and, in addition, interacts with Tyr356, a residue conserved in most class 2 DHODH proteins. Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone undergo changes in conformation upon inhibitor binding. Using our data, the loop regions of residues Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/cofactor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Sitios de Unión/genética , Compuestos de Bifenilo/química , Compuestos de Bifenilo/metabolismo , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Cristalografía por Rayos X , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Mononucleótido de Flavina/metabolismo , Humanos , Naftalenos/química , Naftalenos/metabolismo , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Ácido Orótico/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquinona/metabolismo , ortoaminobenzoatos/síntesis química , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismoRESUMEN
Plasmodium falciparum is the causative agent of the most severe type of malaria, a life-threatening disease affecting the lives of over three billion people. Factors like widespread resistance against available drugs and absence of an effective vaccine are seriously compounding control of the malaria parasite. Thus, there is an urgent need for the identification and validation of new drug targets. The enzymes of the polyamine biosynthesis pathway have been suggested as possible targets for the treatment of malaria. One of these enzymes is spermidine synthase (SPDS, putrescine aminopropyltransferase), which catalyzes the transfer of an aminopropyl moiety from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine. Here we present the three-dimensional structure of P. falciparum spermidine synthase (pfSPDS) in apo form, in complex with dcAdoMet and two inhibitors, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and trans-4-methylcyclohexylamine (4MCHA). The results show that binding of dcAdoMet to pfSPDS stabilizes the conformation of the flexible gatekeeper loop of the enzyme and affects the conformation of the active-site amino acid residues, preparing the protein for binding of the second substrate. The complexes of AdoDATO and 4MCHA with pfSPDS reveal the mode of interactions of these compounds with the enzyme. While AdoDATO essentially fills the entire active-site pocket, 4MCHA only occupies part of it, which suggests that simple modifications of this compound may yield more potent inhibitors of pfSPDS.