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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
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SARS-CoV-2 , Humanos , Regulación Alostérica , Secuencia de Aminoácidos , COVID-19 , Microscopía por Crioelectrón , Endorribonucleasas/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/químicaRESUMEN
l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.
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Ácido Ascórbico , Frutas , Myrtaceae , Proteínas de Plantas , Ácido Ascórbico/metabolismo , Ácido Ascórbico/biosíntesis , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Myrtaceae/metabolismo , Myrtaceae/genética , Galactosa Deshidrogenasas/metabolismo , Galactosa Deshidrogenasas/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genéticaRESUMEN
Frutalin (FTL) is a multiple-binding lectin belonging to the jacalin-related lectin (JRL) family and derived from Artocarpus incisa (breadfruit) seeds. This lectin specifically recognizes and binds α-d-galactose. FTL has been successfully used in immunobiological research for the recognition of cancer-associated oligosaccharides. However, the molecular bases by which FTL promotes these specific activities remain poorly understood. Here, we report the whole 3D structure of FTL for the first time, as determined by X-ray crystallography. The obtained crystals diffracted to 1.81 Å (Apo-frutalin) and 1.65 Å (frutalin-d-Gal complex) of resolution. The lectin exhibits post-translational cleavage yielding an α- (133 amino acids) and ß-chain (20 amino acids), presenting a homotetramer when in solution, with a typical JRL ß-prism. The ß-prism was composed of three 4-stranded ß-sheets forming three antiparallel Greek key motifs. The carbohydrate-binding site (CBS) involved the N-terminus of the α-chain and was formed by four key residues: Gly25, Tyr146, Trp147 and Asp149. Together, these results were used in molecular dynamics simulations in aqueous solutions to shed light on the molecular basis of FTL-ligand binding. The simulations suggest that Thr-Ser-Ser-Asn (TSSN) peptide excision reduces the rigidity of the FTL CBS, increasing the number of interactions with ligands and resulting in multiple-binding sites and anomeric recognition of α-d-galactose sugar moieties. Our findings provide a new perspective to further elucidate the versatility of FTL in many biological activities.
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Artocarpus/química , Galactosa/química , Galectinas/química , Semillas/química , Sitios de Unión , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Septins are GTP-binding proteins that will often spontaneously assemble into filaments. In some species, particularly budding yeast, it is well known that these are capable of associating with membranes in order to fulfill their cellular role as a component of the cytoskeleton. Different from other human septins, SEPT7 appears to be unique in that it is an essential component of all hetero-oligomeric complexes described to date. As a step towards understanding the molecular basis of filament assembly, here we present two high-resolution structures of the SEPT7 GTPase domain complexed with GDP. One of these reveals a previously unreported coordination for the magnesium ion involving four water molecules and only a tenuous connection to the protein. The higher resolution structures provide unambiguous insight into the interactions at the G-interface where a structural motif based on an antiparallel ß-bridge allows for the rationalization of why some septins show nucleotide-dependent ß-strand slippage and others do not.
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Proteínas de Ciclo Celular/química , Proteínas de Unión al GTP/química , Septinas/química , Sitios de Unión , Cristalografía por Rayos X , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Manganeso/química , Conformación Proteica en Lámina beta , Dominios Proteicos , Agua/químicaRESUMEN
The 21st amino acid, selenocysteine (Sec), is synthesized on its dedicated transfer RNA (tRNASec). In bacteria, Sec is synthesized from Ser-tRNA[Ser]Sec by Selenocysteine Synthase (SelA), which is a pivotal enzyme in the biosynthesis of Sec. The structural characterization of bacterial SelA is of paramount importance to decipher its catalytic mechanism and its role in the regulation of the Sec-synthesis pathway. Here, we present a comprehensive single-particle cryo-electron microscopy (SPA cryoEM) structure of the bacterial SelA with an overall resolution of 2.69 Å. Using recombinant Escherichia coli SelA, we purified and prepared samples for single-particle cryoEM. The structural insights from SelA, combined with previous in vivo and in vitro knowledge, underscore the indispensable role of decamerization in SelA's function. Moreover, our structural analysis corroborates previous results that show that SelA adopts a pentamer of dimers configuration, and the active site architecture, substrate binding pocket, and key K295 catalytic residue are identified and described in detail. The differences in protein architecture and substrate coordination between the bacterial enzyme and its counterparts offer compelling structural evidence supporting the independent molecular evolution of the bacterial and archaea/eukarya Ser-Sec biosynthesis present in the natural world.
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Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that C. intestinalis septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had CiSEPT2 occupying the terminal position were able to assemble into filaments via NC-interface association. The CiSEPT7-CiSEPT9 tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7 Å) provides a well-defined view of the central NC-interface. On the other hand, the CiSEPT7-CiSEPT9 G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that C. intestinalis may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.
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Ciona intestinalis , Microscopía por Crioelectrón , Modelos Moleculares , Multimerización de Proteína , Septinas , Ciona intestinalis/metabolismo , Ciona intestinalis/química , Ciona intestinalis/genética , Septinas/metabolismo , Septinas/química , Septinas/genética , Animales , Humanos , Nucleótidos/metabolismo , Nucleótidos/química , Conformación Proteica , Unión ProteicaRESUMEN
In adult schistosomes, the enzyme adenosine kinase (AK) is responsible for the incorporation of some adenosine analogues, such as 2-fluoroadenosine and tubercidin, into the nucleotide pool, but not others. In the present study, the structures of four complexes of Schistosoma mansoni AK bound to adenosine and adenosine analogues are reported which shed light on this observation. Two differences in the adenosine-binding site in comparison with the human counterpart (I38Q and T36A) are responsible for their differential specificities towards adenosine analogues, in which the Schistosoma enzyme does not tolerate bulky substituents at the N7 base position. This aids in explaining experimental data which were reported in the literature more than two decades ago. Furthermore, there appears to be considerable plasticity within the substrate-binding sites that affects the side-chain conformation of Ile38 and causes a previously unobserved flexibility within the loop comprising residues 286-299. These results reveal that the latter can be sterically occluded in the absence of ATP. Overall, these results contribute to the body of knowledge concerning the enzymes of the purine salvage pathway in this important human parasite.
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Adenosina Quinasa/química , Adenosina/química , Schistosoma mansoni/enzimología , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina Quinasa/genética , Adenosina Quinasa/metabolismo , Animales , Cristalización , Cristalografía por Rayos X , Humanos , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Alineación de Secuencia , Especificidad por Sustrato/genéticaRESUMEN
BACKGROUND: Chromobacterium violaceum is a free-living ß-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the ß-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum. RESULTS: The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-ß-D-N,N'-diacetylchitobiose and p-nitrophenyl-ß-D-N,N',N"-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60°C for 30 min. CONCLUSIONS: A C. violaceum chitinase was expressed in E. coli and purified by affinity chromatography on a chitin matrix. The secretion of the recombinant protein into the culture medium was directed by its native signal peptide. The mature enzyme was able to hydrolyze colloidal chitin and synthetic substrates. This newly identified signal peptide is a promising secretion factor that should be further investigated in future studies, aiming to demonstrate its usefulness as an alternative tool for the extracellular production of recombinant proteins in E. coli.
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Quitinasas/biosíntesis , Chromobacterium/enzimología , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Cromatografía de Afinidad , Clonación Molecular , Vectores Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes/biosíntesis , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.
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Hidrolasas , Tereftalatos Polietilenos , Tereftalatos Polietilenos/química , Tereftalatos Polietilenos/metabolismo , Regiones Antárticas , Hidrolasas/química , Hidrólisis , Ingeniería de Proteínas , PlásticosRESUMEN
Schistosomiasis is a parasitic infection caused by trematode worms (also called blood flukes) of the genus Schistosoma sp., which affects over 230 million people worldwide, causing 200,000 deaths annually. There is no vaccine or new drugs available, which represents a worrying aspect, since there is loss of sensitivity of the parasite to the medication recommended by the World Health Organization, Praziquantel. The present study evaluated the effects of the recombinant enzymes of S. mansoni Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT), Purine Nucleoside Phosphorylase (PNP) and the MIX of both enzymes in the immunotherapy of schistosomiasis in murine model. These enzymes are part of the purine salvage pathway, the only metabolic pathway present in the parasite for this purpose, being essential for the synthesis of DNA and RNA. Female mice of Swiss and BALB/c strains were infected with cercariae and treated, intraperitoneally, with three doses of 100 µg of enzymes. After the immunotherapy, the eggs and adult worms were counted in the feces; the number of eosinophils from the fluid in the peritoneal cavity and peripheral blood was observed; and the quantification of the cytokine IL-4 and the production of antibodies IgE was analyzed. The evaluation of the number of granulomas and collagen deposition via histological slides of the liver was performed. The results demonstrate that immunotherapy with the enzyme HGPRT seems to stimulate the production of IL-4 and promoted a significant reduction of granulomas in the liver in treated animals. The treatment with the enzyme PNP and the MIX was able to reduce the number of worms in the liver and in the mesenteric vessels of the intestine, to reduce the number of eggs in the feces and to negatively modulate the number of eosinophils. Therefore, immunotherapy with the recombinant enzymes of S. mansoni HGPRT and PNP might contribute to the control and reduction of the pathophysiological aspects of schistosomiasis, helping to decrease the morbidity associated with the infection in murine model.
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Schistosomiasis is a parasitic disease that is part of the neglected tropical diseases (NTDs), which cause significant levels of morbidity and mortality in millions of people throughout the world. The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) represents a potential target for discovering new agents, and neolignans stand out as an important class of compounds. In this work, molecular modeling studies and biological assays of a set of neolignans were conducted against the PNP enzymes of the parasite and the human homologue (HssPNP). The results of the molecular docking described that the neolignans showed good complementarity by the active site of SmPNP. Molecular dynamics (MD) studies revealed that both complexes (Sm/HssPNP - neolignan compounds) were stable by analyzing the root mean square deviation (RMSD) values, and the binding free energy values suggest that the selected structures can interact and inhibit the catalytic activity of the SmPNP. Finally, the biological assay indicated that the selected neolignans presented a better molecular profile of inhibition compared to the human enzyme, as these ligands did not have the capacity to inhibit enzymatic activity, indicating that these compounds are promising candidates and that they can be used in future research in chemotherapy for schistosomiasis.Communicated by Ramaswamy H. Sarma.
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Lignanos , Esquistosomiasis , Animales , Inhibidores Enzimáticos/química , Humanos , Simulación del Acoplamiento Molecular , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Schistosoma mansoni/metabolismoRESUMEN
In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 Å and a local resolution of between ~3.0 Å and ~5.0 Å. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.
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Proteínas de Ciclo Celular/química , Septinas/química , Proteínas de Ciclo Celular/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Unión Proteica , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Septinas/metabolismoRESUMEN
SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.
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[This corrects the article DOI: 10.1371/journal.pone.0209988.].
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Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5', the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.
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Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.
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Adenilosuccinato Liasa/inmunología , Antígenos Helmínticos/inmunología , Nucleósido-Difosfato Quinasa/inmunología , Schistosoma mansoni/enzimología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/parasitología , Animales , Antígenos Helmínticos/administración & dosificación , Biomarcadores , Citocinas/sangre , Eosinófilos , Femenino , Inmunización , Esquemas de Inmunización , Recuento de Leucocitos , Hígado/metabolismo , Hígado/parasitología , Hígado/patología , Ratones , Carga de Parásitos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Esquistosomiasis mansoni/patología , Esquistosomiasis mansoni/prevención & controlRESUMEN
Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.
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Liasas/metabolismo , Fosfotransferasas/metabolismo , Selenocisteína/biosíntesis , Selenoproteínas/metabolismo , Trypanosoma brucei brucei/enzimología , Conformación Proteica , Proteínas Protozoarias/metabolismo , Selenio/metabolismoRESUMEN
Citrus canker is a plant disease caused by the bacteria Xanthomonas citri subsp. citri that affects all domestic varieties of citrus. Some annotated genes from the X. citri subsp. citri genome are assigned to an interesting class named "pathogenicity, virulence and adaptation". Amongst these is sodM, which encodes for the gene product XcSOD, one of four superoxide dismutase homologs predicted from the genome. SODs are widespread enzymes that play roles in the oxidative stress response, catalyzing the degradation of the deleterious superoxide radical. In Xanthomonas, SOD has been associated with pathogenesis as a counter measure against the plant defense response. In this work we initially present the 1.8 Å crystal structure of XcSOD, a manganese containing superoxide dismutase from Xanthomonas citri subsp. citri. The structure bears all the hallmarks of a dimeric member of the MnSOD family, including the conserved hydrogen-bonding network residues. Despite the apparent gene redundancy, several attempts to obtain a sodM deletion mutant were unsuccessful, suggesting the encoded protein to be essential for bacterial survival. This intriguing observation led us to extend our structural studies to the remaining three SOD homologs, for which comparative models were built. The models imply that X. citri subsp. citri produces an iron-containing SOD which is unlikely to be catalytically active along with two conventional Cu,ZnSODs. Although the latter are expected to possess catalytic activity, we propose they may not be able to replace XcSOD for reasons such as distinct subcellular compartmentalization or differential gene expression in pathogenicity-inducing conditions.
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Proteínas Bacterianas/química , Superóxido Dismutasa/química , Xanthomonas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Genes Esenciales , Modelos Moleculares , Conformación Proteica , Superóxido Dismutasa/genética , Xanthomonas/genética , Xanthomonas/patogenicidadRESUMEN
Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.
Asunto(s)
Proteínas del Helminto/química , Proteínas del Helminto/genética , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/genética , Isoenzimas/química , Isoenzimas/genética , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Proteínas del Helminto/metabolismo , Hipoxantina Fosforribosiltransferasa/metabolismo , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Reproducción , Schistosoma mansoni/química , Schistosoma mansoni/genética , Schistosoma mansoni/fisiología , Alineación de SecuenciaRESUMEN
Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 µM for cytosine, and a KM of 76.3 µM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.