RESUMEN
RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.
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Microscopía por Crioelectrón , Glicina Hidroximetiltransferasa , Glicina Hidroximetiltransferasa/metabolismo , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/química , Humanos , ARN/metabolismo , ARN/genética , Serina/metabolismo , Regulación Alostérica , Unión Proteica , Filogenia , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Glicina/metabolismo , Glicina/química , Sitios de UniónRESUMEN
BACKGROUND: Marinesco-Sjögren syndrome (MSS) is an autosomal recessive neuromuscular disorder that arises in early childhood and is characterized by congenital cataracts, myopathy associated with muscle weakness, and degeneration of Purkinje neurons leading to ataxia. About 60% of MSS patients have loss-of-function mutations in the SIL1 gene. Sil1 is an endoplasmic reticulum (ER) protein required for the release of ADP from the master chaperone Bip, which in turn will release the folded proteins. The expression of non-functional Sil1 leads to the accumulation of unfolded proteins in the ER and this triggers the unfolded protein response (UPR). A dysfunctional UPR could be a key element in the pathogenesis of MSS, although our knowledge of the molecular pathology of MSS is still incomplete. METHODS: RNA-Seq transcriptomics was analysed using the String database and the Ingenuity Pathway Analysis platform. Fluorescence confocal microscopy was used to study the remodelling of the extracellular matrix (ECM). Transmission electron microscopy (TEM) was used to reveal the morphology of the ECM in vitro and in mouse tendon. RESULTS: Our transcriptomic analysis, performed on patient-derived fibroblasts, revealed 664 differentially expressed (DE) transcripts. Enrichment analysis of DE genes confirmed that the patient fibroblasts have a membrane trafficking issue. Furthermore, this analysis indicated that the extracellular space/ECM and the cell adhesion machinery, which together account for around 300 transcripts, could be affected in MSS. Functional assays showed that patient fibroblasts have a reduced capacity of ECM remodelling, reduced motility, and slower spreading during adhesion to Petri dishes. TEM micrographs of negative-stained ECM samples from these fibroblasts show differences of filaments in terms of morphology and size. Finally, structural analysis of the myotendinous junction of the soleus muscle and surrounding regions of the Achilles tendon revealed a disorganization of collagen fibres in the mouse model of MSS (woozy). CONCLUSIONS: ECM alterations can affect the proper functioning of several organs, including those damaged in MSS such as the central nervous system, skeletal muscle, bone and lens. On this basis, we propose that aberrant ECM is a key pathological feature of MSS and may help explain most of its clinical manifestations.
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Matriz Extracelular , Fibroblastos , Degeneraciones Espinocerebelosas , Tendones , Fibroblastos/metabolismo , Fibroblastos/patología , Matriz Extracelular/metabolismo , Humanos , Animales , Tendones/patología , Tendones/metabolismo , Degeneraciones Espinocerebelosas/patología , Degeneraciones Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/metabolismo , Respuesta de Proteína Desplegada , Ratones , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Perfilación de la Expresión GénicaRESUMEN
BACKGROUND: The underlying mechanism of Parkinson's disease are still unidentified, but excitotoxicity, oxidative stress, and neuroinflammation are considered key actors. Proliferator activated receptors (PPARs) are transcription factors involved in the control of numerous pathways. Specifically, PPARß/δ is recognized as an oxidative stress sensor, and we have previously reported that it plays a detrimental role in neurodegeneration. METHODS: Basing on this concept, in this work, we tested the potential effects of a specific PPARß/δ antagonist (GSK0660) in an in vitro model of Parkinson's disease. Specifically, live-cell imaging, gene expression, Western blot, proteasome analyses, mitochondrial and bioenergetic studies were performed. Since we obtained promising results, we tested this antagonist in a 6-hydroxydopamine hemilesioned mouse model. In the animal model, behavioral tests, histological analysis, immunofluorescence and western blot of substantia nigra and striatum upon GSK0660 were assayed. RESULTS: Our findings suggested that PPARß/δ antagonist has neuroprotective potential due to neurotrophic support, anti-apoptotic and anti-oxidative effects paralleled to an amelioration of mitochondria and proteasome activity. These findings are strongly supported also by the siRNA results demonstrating that by silencing PPARß/δ a significative rescue of the dopaminergic neurons was obtained, thus indicating an involvement of PPARß/δ in PD's pathogenesis. Interestingly, in the animal model, GSK0660 treatment confirmed neuroprotective effects observed in the in vitro studies. Neuroprotective effects were highlighted by the behavioural performance and apomorphine rotation tests amelioration and the reduction of dopaminergic neuronal loss. These data were also confirmed by imaging and western blotting, indeed, the tested compound decreased astrogliosis and activated microglia, concomitant with an upregulation of neuroprotective pathways. CONCLUSIONS: In summary, PPARß/δ antagonist displayed neuroprotective activities against 6-hydroxydopamine detrimental effects both in vitro and in vivo models of Parkinson's disease, suggesting that it may represent a novel therapeutic approach for this disorder.
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Fármacos Neuroprotectores , PPAR-beta , Enfermedad de Parkinson , Animales , Ratones , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Oxidopamina , Complejo de la Endopetidasa ProteasomalRESUMEN
The demonstration of the first enzyme-based electrode to detect glucose, published in 1967 by S. J. Updike and G. P. Hicks, kicked off huge efforts in building sensors where biomolecules are exploited as native or modified to achieve new or improved sensing performances. In this growing area, bionanotechnology has become prominent in demonstrating how nanomaterials can be tailored into responsive nanostructures using biomolecules and integrated into sensors to detect different analytes, e.g., biomarkers, antibiotics, toxins and organic compounds as well as whole cells and microorganisms with very high sensitivity. Accounting for the natural affinity between biomolecules and almost every type of nanomaterials and taking advantage of well-known crosslinking strategies to stabilize the resulting hybrid nanostructures, biosensors with broad applications and with unprecedented low detection limits have been realized. This review depicts a comprehensive collection of the most recent biochemical and biophysical strategies for building hybrid devices based on bioconjugated nanomaterials and their applications in label-free detection for diagnostics, food and environmental analysis.
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Técnicas Biosensibles , Nanoestructuras , Nanoestructuras/química , Técnicas Biosensibles/métodos , BiomarcadoresRESUMEN
Affibodies and designed ankyrin repeat proteins (DARPins) are synthetic proteins originally derived from the Staphylococcus aureus virulence factor protein A and the human ankyrin repeat proteins, respectively. The use of these molecules in healthcare has been recently proposed as they are endowed with biochemical and biophysical features heavily demanded to target and fight diseases, as they have a strong binding affinity, solubility, small size, multiple functionalization sites, biocompatibility, and are easy to produce; furthermore, impressive chemical and thermal stability can be achieved. especially when using affibodies. In this sense, several examples reporting on affibodies and DARPins conjugated to nanomaterials have been published, demonstrating their suitability and feasibility in nanomedicine for cancer therapy. This minireview provides a survey of the most recent studies describing affibody- and DARPin-conjugated zero-dimensional nanomaterials, including inorganic, organic, and biological nanoparticles, nanorods, quantum dots, liposomes, and protein- and DNA-based assemblies for targeted cancer therapy in vitro and in vivo.
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Nanoestructuras , Neoplasias , Humanos , Proteínas de Repetición de Anquirina Diseñadas , Proteínas/química , Neoplasias/tratamiento farmacológicoRESUMEN
In this study, nitrogen and sulfur co-doped carbon dots (NS-CDs) were investigated for the detection of heavy metals in water through absorption-based colorimetric response. NS-CDs were synthesized by a simple one-pot hydrothermal method and characterized by TEM, STEM-coupled with energy dispersive X-ray analysis, NMR, and IR spectroscopy. Addition of Cu(II) ions to NS-CD aqueous solutions gave origin to a distinct absorption band at 660 nm which was attributed to the formation of cuprammonium complexes through coordination with amino functional groups of NS-CDs. Absorbance increased linearly with Cu(II) concentration in the range 1-100 µM and enabled a limit of detection of 200 nM. No response was observed with the other tested metals, including Fe(III) which, however, appreciably decreased sensitivity to copper. Increase of pH of the NS-CD solution up to 9.5 greatly reduced this interference effect and enhanced the response to Cu(II), thus confirming the different nature of the two interactions. In addition, a concurrent response to Co(II) appeared in a different spectral region, thus suggesting the possibility of dual-species multiple sensitivity. The present method neither requires any other reagents nor any previous assay treatment and thus can be a promising candidate for low-cost monitoring of copper onsite and by unskilled personnel.
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Carbono , Puntos Cuánticos , Carbono/química , Cobalto , Colorimetría/métodos , Cobre/análisis , Compuestos Férricos , Nitrógeno/química , Puntos Cuánticos/química , Azufre , AguaRESUMEN
Morpheeins are proteins that reversibly assemble into different oligomers, whose architectures are governed by conformational changes of the subunits. This property could be utilized in bionanotechnology where the building of nanometric and new high-ordered structures is required. By capitalizing on the adaptability of morpheeins to create patterned structures and exploiting their inborn affinity toward inorganic and living matter, "bottom-up" creation of nanostructures could be achieved using a single protein building block, which may be useful as such or as scaffolds for more complex materials. Peroxiredoxins represent the paradigm of a morpheein that can be applied to bionanotechnology. This review describes the structural and functional transitions that peroxiredoxins undergo to form high-order oligomers, e.g., rings, tubes, particles, and catenanes, and reports on the chemical and genetic engineering approaches to employ them in the generation of responsive nanostructures and nanodevices. The usefulness of the morpheeins' behavior is emphasized, supporting their use in future applications.
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Nanoestructuras/química , Peroxirredoxinas/química , Proteínas/química , Biopolímeros/química , Peroxirredoxinas/metabolismo , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
Delivery of molecules into intracellular compartments is one of the fundamental requirements in molecular biology. However, the possibility of delivering a precise number of nano-objects with single-particle resolution is still an open challenge. Here we present an electrophoretic platform based on 3D hollow nanoelectrodes to enable delivery of single nanoparticles into single selected cells and monitoring of the single-particle delivery by surface-enhanced Raman scattering (SERS). The gold-coated hollow nanoelectrode capable of confinement and enhancement of electromagnetic fields upon laser illumination can distinguish the SERS signals of a single nanoparticle flowing through the nanoelectrode. Tight wrapping of cell membranes around the nanoelectrodes allows effective membrane electroporation such that single gold nanorods are delivered on demand into a living cell by electrophoresis. The capability of the 3D hollow nanoelectrodes to porate cells and reveal single emitters from the background in continuous flow is promising for the analysis of both intracellular delivery and sampling.
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In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells. By providing a selective targeting mechanism for cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the chemistry of ADC linker conjugation together with strategies adopted to improve antibody tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin (Ig) genes and antibody humanization have greatly reduced some of the problems of the first generation of ADCs, beset by problems, such as random coupling of the payload and immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and will likely prove an important modality for the treatment of cancer in the near future.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Anticuerpos Monoclonales/inmunología , Antineoplásicos/inmunología , Humanos , Inmunoconjugados/inmunología , Neoplasias/inmunología , Neoplasias/patologíaRESUMEN
BACKGROUND: The ferroxidase center of DNA-binding protein from starved cells (Dps) is a major player in the iron oxidation/detoxification process that leads to a decreased reactive oxygen species production. The possible Mn(II) participation in this process has been studied in Dps from Kineococcus radiotolerans, a radiation-resistant bacterium with a high cytosolic Mn/Fe ratio and a high capacity to survive ionizing and stress conditions. METHODS: The X-ray structure of recombinant K. radiotolerans Dps loaded with Mn(II) has been solved at 2.0Å resolution. Mn(II) binding to K. radiotolerans Dps and its effect on Fe(II) oxidation have been characterized in spectroscopic measurements. RESULTS: In K. radiotolerans Dps, the Fe-Fe ferroxidase center can have a Mn-Fe composition. Mn(II) binds only at the high affinity, so-called A site, whereas Fe(II) binds also at the low affinity, so-called B site. The Mn-Fe and Fe-Fe centers behave distinctly upon iron oxidation by O2. A site-bound Mn(II) or Fe(II) plays a catalytic role, while B site-bound Fe(II) behaves like a substrate and can be replaced by another Fe(II) after oxidation. When H2O2 is the Fe(II) oxidant, single electrons are transferred to aromatic residues near the ferroxidase center and give rise to intra-protein radicals thereby limiting OH release in solution. The presence of the Mn-Fe center results in significant differences in the development of such intra-protein radicals. CONCLUSIONS: Mn(II) bound at the Dps ferroxidase center A site undergoes redox cycling provided the B site contains Fe. GENERAL SIGNIFICANCE: The results provide a likely molecular mechanism for the protective role of Mn(II) under oxidative stress conditions as it participates in redox cycling in the hetero-binuclear ferroxidase center.
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Actinomycetales/enzimología , Proteínas Bacterianas/química , Ceruloplasmina/química , Proteínas de Unión al ADN/química , Estrés Oxidativo/fisiología , Actinomycetales/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Radical Hidroxilo/química , Radical Hidroxilo/metabolismo , Hierro/química , Hierro/metabolismo , Manganeso/química , Manganeso/metabolismo , Estructura Cuaternaria de ProteínaRESUMEN
Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.
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Esquistosomiasis , Esquistosomicidas , Animales , Ratones , Esquistosomicidas/farmacología , Esquistosomicidas/uso terapéutico , Praziquantel/farmacología , Schistosoma , NADH NADPH Oxidorreductasas/farmacología , NADH NADPH Oxidorreductasas/uso terapéutico , Schistosoma mansoniRESUMEN
Suicide gene therapy is a relatively novel form of cancer therapy in which a gene coding for enzymes or protein toxins is delivered through targeting systems such as vesicles, nanoparticles, peptide or lipidic co-adjuvants. The use of toxin genes is particularly interesting since their catalytic activity can induce cell death, damaging in most cases the translation machinery (ribosomes or protein factors involved in protein synthesis) of quiescent or proliferating cells. Thus, toxin gene delivery appears to be a promising tool in fighting cancer. In this review we will give an overview, describing some of the bacterial and plant enzymes studied so far for their delivery and controlled expression in tumor models.
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Inmunotoxinas , Neoplasias , Toxinas Biológicas , Terapia Genética , Humanos , Inmunotoxinas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Ribosomas/metabolismo , Toxinas Biológicas/metabolismoRESUMEN
Enzymes in the thiol redox systems of microbial pathogens are promising targets for drug development. In this study we characterized the thioredoxin reductase (TrxR) selenoproteins from Brugia malayi and Onchocerca volvulus, filarial nematode parasites and causative agents of lymphatic filariasis and onchocerciasis, respectively. The two filarial enzymes showed similar turnover numbers and affinities for different thioredoxin (Trx) proteins, but with a clear preference for the autologous Trx. Human TrxR1 (hTrxR1) had a high and similar specific activity versus the human and filarial Trxs, suggesting that, in vivo, hTrxR1 could possibly be the reducing agent of parasite Trxs once they are released into the host. Both filarial TrxRs were efficiently inhibited by auranofin and by a recently described inhibitor of human TrxR1 (TRi-1), but not as efficiently by the alternative compound TRi-2. The enzyme from B. malayi was structurally characterized also in complex with NADPH and auranofin, producing the first crystallographic structure of a nematode TrxR. The protein represents an unusual fusion of a mammalian-type TrxR protein architecture with an N-terminal glutaredoxin-like (Grx) domain lacking typical Grx motifs. Unlike thioredoxin glutathione reductases (TGRs) found in platyhelminths and mammals, which are also Grx-TrxR domain fusion proteins, the TrxRs from the filarial nematodes lacked glutathione disulfide reductase and Grx activities. The structural determinations revealed that the Grx domain of TrxR from B. malayi contains a cysteine (C22), conserved in TrxRs from clade IIIc nematodes, that directly interacts with the C-terminal cysteine-selenocysteine motif of the homo-dimeric subunit. Interestingly, despite this finding we found that altering C22 by mutation to serine did not affect enzyme catalysis. Thus, although the function of the Grx domain in these filarial TrxRs remains to be determined, the results obtained provide insights on key properties of this important family of selenoprotein flavoenzymes that are potential drug targets for treatment of filariasis.
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Brugia Malayi , Onchocerca volvulus , Parásitos , Animales , Auranofina/farmacología , Brugia Malayi/metabolismo , Cisteína/metabolismo , Humanos , Mamíferos/metabolismo , Onchocerca volvulus/genética , Onchocerca volvulus/metabolismo , Oxidación-Reducción , Parásitos/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Numerous studies have shown a strong correlation between the number of neurofibrillary tangles of the tau protein and Alzheimer's disease progression, making the quantitative detection of tau very promising from a clinical point of view. However, the lack of highly reliable fluorescent probes for selective imaging of tau neurofibrillary tangles is a major challenge due to sharing similar ß-sheet motifs with homologous Amyloid-ß fibrils. In the current work, we describe the rational design and the in silico evaluation of a small-size focused library of fluorescent probes, consisting of a BODIPY core (electron acceptor) featuring highly conjugated systems (electron donor) with a length in the range 13-19 Å at C3. Among the most promising probes in terms of binding mode, theoretical affinity and polarity, BT1 has been synthesized and tested in vitro onto human induced pluripotent stem cells derived neuronal cell cultures. The probe showed excellent photophysical properties and high selectivity allowing in vitro imaging of hyperphosphorylated tau protein filaments with minimal background noise. Our findings offer new insight into the structure-activity relationship of this class of tau selective fluorophores, paving the way for boosting tau tangle detection in patients possibly through retinal spectral scans.
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Células Madre Pluripotentes Inducidas , Compuestos de Boro , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Proteínas tau/metabolismoRESUMEN
A novel suicide gene therapy approach was tested in U87 MG glioblastoma multiforme cells. A 26nt G-rich double-stranded DNA aptamer (AS1411) was integrated into a vector at the 5' of a mammalian codon-optimized saporin gene, under CMV promoter. With this plasmid termed "APTSAP", the gene encoding ribosome-inactivating protein saporin is driven intracellularly by the glioma-specific aptamer that binds to cell surface-exposed nucleolin and efficiently kills target cells, more effectively as a polyethyleneimine (PEI)-polyplex. Cells that do not expose nucleolin at the cell surface such as 3T3 cells, used as a control, remain unaffected. Suicide gene-induced cell killing was not observed when the inactive saporin mutant SAPKQ DNA was used in the (PEI)-polyplex, indicating that saporin catalytic activity mediates the cytotoxic effect. Rather than apoptosis, cell death has features resembling autophagic or methuosis-like mechanisms. These main findings support the proof-of-concept of using PEI-polyplexed APTSAP for local delivery in rat glioblastoma models.
RESUMEN
Fragment screening is a powerful drug discovery approach particularly useful for enzymes difficult to inhibit selectively, such as the thiol/selenol-dependent thioredoxin reductases (TrxRs), which are essential and druggable in several infectious diseases. Several known inhibitors are reactive electrophiles targeting the selenocysteine-containing C-terminus and thus often suffering from off-target reactivity in vivo. The lack of structural information on the interaction modalities of the C-terminus-targeting inhibitors, due to the high mobility of this domain and the lack of alternative druggable sites, prevents the development of selective inhibitors for TrxRs. In this work, fragments selected from actives identified in a large screen carried out against Thioredoxin Glutathione Reductase from Schistosoma mansoni (SmTGR) were probed by X-ray crystallography. SmTGR is one of the most promising drug targets for schistosomiasis, a devastating, neglected disease. Utilizing a multicrystal method to analyze electron density maps, structural analysis, and functional studies, three binding sites were characterized in SmTGR: two sites are close to or partially superposable with the NADPH binding site, while the third one is found between two symmetry related SmTGR subunits of the crystal lattice. Surprisingly, one compound bound to this latter site stabilizes, through allosteric effects mediated by the so-called guiding bar residues, the crucial redox active C-terminus of SmTGR, making it finally visible at high resolution. These results further promote fragments as small molecule probes for investigating functional aspects of the target protein, exemplified by the allosteric effect on the C-terminus, and providing fundamental chemical information exploitable in drug discovery.
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Antiparasitarios/química , Schistosoma mansoni/efectos de los fármacos , Animales , Complejos Multienzimáticos , NADH NADPH Oxidorreductasas/genéticaRESUMEN
Selective suicide inhibitors represent a seductively attractive approach for inactivation of therapeutically relevant enzymes since they are generally devoid of off-target toxicity in vivo. While most suicide inhibitors are converted to reactive species at enzyme active sites, theoretically bioactivation can also occur in ectopic (secondary) sites that have no known function. Here, we report an example of such an "ectopic suicide inhibition", an unprecedented bioactivation mechanism of a suicide inhibitor carried out by a non-catalytic site of thioredoxin glutathione reductase (TGR). TGR is a promising drug target to treat schistosomiasis, a devastating human parasitic disease. Utilizing hits selected from a high throughput screening campaign, time-resolved X-ray crystallography, molecular dynamics, mass spectrometry, molecular modeling, protein mutagenesis and functional studies, we find that 2-naphtholmethylamino derivatives bound to this novel ectopic site of Schistosoma mansoni (Sm)TGR are transformed to covalent modifiers and react with its mobile selenocysteine-containing C-terminal arm. In particular, one 2-naphtholmethylamino compound is able to specifically induce the pro-oxidant activity in the inhibited enzyme. Since some 2-naphtholmethylamino analogues show worm killing activity and the ectopic site is not conserved in human orthologues, a general approach to development of novel and selective anti-parasitic therapeutics against schistosoma is proposed.
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Complejos Multienzimáticos , NADH NADPH Oxidorreductasas , Animales , Cristalografía por Rayos X , Glutatión Reductasa , Humanos , NADH NADPH Oxidorreductasas/genética , Schistosoma mansoni , Reductasa de Tiorredoxina-DisulfuroRESUMEN
BACKGROUND: Proteins are efficient supramolecular scaffolds to drive self-assembly of nanomaterials into regular colloidal structures suitable for several purposes, including cell imaging and drug delivery. Proteins, in particular, can bind to gold nanoparticles (AuNPs) through van der Waals and electrostatic forces as well as coordination and hydrogen bonds leading their assembly into responsive nanostructures. METHODS: Bioconjugation of alkyne Raman tag-labeled 20 nm AuNPs with the ring-shaped protein Peroxiredoxin (Prx), characterized by a symmetric homo-oligomeric circular arrangement, has been investigated by absorption spectroscopy, transmission and scanning electron microscopy. The plasmonic behavior of the resulting hybrid assemblies has been assessed by Surface Enhanced Raman Scattering (SERS). RESULTS: The ring-shaped Prx molecules are demonstrated to adsorb onto the gold surface acting as "sticky" bio-linkers between adjacent nanoparticles to drive self-assembly into small colloidal AuNPs arrays. The arrays show nanometric interparticle gaps tailored by the protein ring thickness. The arrays exhibit improved optical activity due to SERS allowing detection of the Raman signals from both the protein and alkyne molecules. CONCLUSIONS: This method can be used to build up SERS-active nanostructures using Prx as both a bio-linker and platform for attaching dyes, two-dimensional materials, such as graphene, and other biomolecules including DNA and enzymes. GENERAL SIGNIFICANCE: The development of colloidal SERS nanostructures is considered a significant step forward in spectroscopic bioanalysis. Though protein-tailored nanofabrication is in a childhood stage, these results demonstrate the versatility of supramolecular proteins as tools to build-up nanostructures which are still impractical to obtain through top-down techniques.
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Oro/química , Nanopartículas del Metal/química , Peroxirredoxinas/química , Adsorción , Alquinos/química , Animales , Modelos Moleculares , Tamaño de la Partícula , Peroxirredoxinas/metabolismo , Schistosoma mansoni/enzimología , Espectrometría Raman , Propiedades de SuperficieRESUMEN
Here, we propose an easy and robust strategy for the versatile preparation of hybrid plasmonic nanopores by means of controlled deposition of single flakes of MoS2 directly on top of metallic holes. The device is realized on silicon nitride membranes and can be further refined by TEM or FIB milling to achieve the passing of molecules or nanometric particles through a pore. Importantly, we show that the plasmonic enhancement provided by the nanohole is strongly accumulated in the 2D nanopore, thus representing an ideal system for single-molecule sensing and sequencing in a flow-through configuration. Here, we also demonstrate that the prepared 2D material can be decorated with metallic nanoparticles that can couple their resonance with the nanopore resonance to further enhance the electromagnetic field confinement at the nanoscale level. This method can be applied to any gold nanopore with a high level of reproducibility and parallelization; hence, it can pave the way to the next generation of solid-state nanopores with plasmonic functionalities. Moreover, the controlled/ordered integration of 2D materials on plasmonic nanostructures opens a pathway towards new investigation of the following: enhanced light emission; strong coupling from plasmonic hybrid structures; hot electron generation; and sensors in general based on 2D materials.
RESUMEN
BACKGROUND: The underlying mechanism of Parkinson's disease are still unidentified, but excitotoxicity, oxidative stress, and neuroinflammation are considered key actors. Proliferator activated receptors (PPARs) are transcription factors involved in the control of numerous pathways. Specifically, PPARß/δ is recognized as an oxidative stress sensor, and we have previously reported that it plays a detrimental role in neurodegeneration. METHODS: Basing on this concept, in this work, we tested the potential effects of a specific PPARß/δ antagonist (GSK0660) in an in vitro model of Parkinson's disease. Specifically, live-cell imaging, gene expression, Western blot, proteasome analyses, mitochondrial and bioenergetic studies were performed. Since we obtained promising results, we tested this antagonist in a 6-hydroxydopamine hemilesioned mouse model. In the animal model, behavioral tests, histological analysis, immunofluorescence and western blot of substantia nigra and striatum upon GSK0660 were assayed. RESULTS: Our findings suggested that PPARß/δ antagonist has neuroprotective potential due to neurotrophic support, anti-apoptotic and anti-oxidative effects paralleled to an amelioration of mitochondria and proteasome activity. These findings are strongly supported also by the siRNA results demonstrating that by silencing PPARß/δ a significative rescue of the dopaminergic neurons was obtained, thus indicating an involvement of PPARß/δ in PD's pathogenesis. Interestingly, in the animal model, GSK0660 treatment confirmed neuroprotective effects observed in the in vitro studies. Neuroprotective effects were highlighted by the behavioural performance and apomorphine rotation tests amelioration and the reduction of dopaminergic neuronal loss. These data were also confirmed by imaging and western blotting, indeed, the tested compound decreased astrogliosis and activated microglia, concomitant with an upregulation of neuroprotective pathways. CONCLUSIONS: In summary, PPARß/δ antagonist displayed neuroprotective activities against 6-hydroxydopamine detrimental effects both in vitro and in vivo models of Parkinson's disease, suggesting that it may represent a novel therapeutic approach for this disorder.