RESUMEN

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).

Asunto(s)

Clostridium botulinum , Flagelina , Glicosiltransferasas , Especificidad por Sustrato , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/química , Flagelina/metabolismo , Flagelina/genética , Flagelina/química , Clostridium botulinum/enzimología , Clostridium botulinum/metabolismo , Clostridium botulinum/genética , Glicosilación , Escherichia coli/genética , Escherichia coli/metabolismo , Azúcares Ácidos/metabolismo , Ingeniería de Proteínas , Ácido N-Acetilneuramínico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Ácidos Siálicos

RESUMEN

By-products like CO2 and organic acids, produced during Clostridium botulinum growth, appear to inhibit its development and reduce ATP production. A decrease in ATP production creates an imbalance in the ATP/GTP ratio. GTP activates CodY, which regulates BoNT expression. This toxin is released into the extracellular medium. Its light chains act as a specific endopeptidase, targeting SNARE proteins. The specific amino acids released enter the cells and are metabolized by the Stickland reaction, resulting in the synthesis of ATP. This ATP might then be used by histidine kinases to activate Spo0A, the main regulator initiating sporulation, through phosphorylation.

Asunto(s)

Toxinas Botulínicas , Clostridium botulinum , Endopeptidasas , Clostridium botulinum/metabolismo , Clostridium botulinum/enzimología , Toxinas Botulínicas/metabolismo , Endopeptidasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Trifosfato/metabolismo , Esporas Bacterianas/metabolismo , Esporas Bacterianas/crecimiento & desarrollo

RESUMEN

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.

Asunto(s)

Toxinas Botulínicas Tipo A , Clostridium botulinum , Péptido Hidrolasas , Ingeniería de Proteínas , Toxinas Botulínicas Tipo A/química , Catálisis , Dominio Catalítico , Clostridium botulinum/enzimología , Clostridium botulinum/metabolismo , Ingeniería de Proteínas/métodos , Especificidad por Sustrato

RESUMEN

Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an effective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (HC/A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant HC/A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand-receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rHC/A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rHC/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 ≈ 27 nM) > FGFR2b (EC50 ≈ 70 nM) > FGFR1c (EC50 ≈ 163 nM); rHC/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 ≈ 18 nM). Mutating the ganglioside binding site in HC/A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein HC/A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization.

Asunto(s)

Toxinas Botulínicas Tipo A/metabolismo , Clostridium botulinum/enzimología , Neurotoxinas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Serogrupo , Transducción de Señal/genética , Animales , Sitios de Unión , Toxinas Botulínicas Tipo A/química , Membrana Celular/metabolismo , Dimerización , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gangliósidos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neurotoxinas/química , Células PC12 , Unión Proteica , Dominios Proteicos , Ratas , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transfección

RESUMEN

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

Asunto(s)

Proteínas Bacterianas/metabolismo , Clostridium botulinum/enzimología , Ribonucleótido Reductasas/metabolismo , Proteínas Bacterianas/clasificación , Dominio Catalítico , Cristalografía por Rayos X , Nucleótidos de Desoxiadenina/química , Dimerización , Escherichia coli/metabolismo , Filogenia , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ribonucleótido Reductasas/clasificación

RESUMEN

Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.

Asunto(s)

Clostridium botulinum/enzimología , Hidroliasas/química , Hidroliasas/metabolismo , Ácido Pirúvico/metabolismo , Alquilación , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clostridium botulinum/química , Cristalografía por Rayos X , Cisteína/química , Estabilidad de Enzimas , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos X

RESUMEN

The extreme toxicity of botulinum neurotoxins (BoNTs) relies on their specific cleavage of SNARE proteins, which eventually leads to muscle paralysis. One newly identified mosaic toxin, BoNT/HA (aka H or FA), cleaves VAMP-2 at a unique position between residues L54 and E55, but the molecular basis underlying VAMP-2 recognition of BoNT/HA remains poorly characterized. Here, we report a ∼2.09 Šresolution crystal structure of the light chain protease domain of BoNT/HA (LC/HA). Structural comparison between LC/HA and LC of BoNT/F1 (LC/F1) reveals distinctive hydrophobic and electrostatic features near the active sites, which may explain their different VAMP-2 cleavage sites. When compared to BoNT/F5 that cleaves VAMP-2 at the same site as BoNT/HA, LC/HA displays higher affinity for VAMP-2, which could be caused by their different surface charge properties surrounding a VAMP-2 exosite-binding cleft. Furthermore, systematic mutagenesis studies on VAMP-2 and structural modeling demonstrate that residues R47 to K59 spanning the cleavage site in VAMP-2 may adopt a novel extended conformation when interacting with LC/HA and LC/F5. Taken together, our structure provides new insights into substrate recognition of BoNT/HA and paves the way for rational design of small molecule or peptide inhibitors against LC/HA.

Asunto(s)

Toxinas Botulínicas Tipo A/química , Clostridium botulinum/química , Proteína 2 de Membrana Asociada a Vesículas/química , Secuencia de Aminoácidos , Sitios de Unión , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/metabolismo , Clonación Molecular , Clostridium botulinum/enzimología , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagénesis , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Especificidad por Sustrato , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo

RESUMEN

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 Å X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells.

Asunto(s)

Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/metabolismo , Dominio Catalítico , Clostridium botulinum/enzimología , Sitios de Unión , Activación Enzimática , Modelos Moleculares , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Botulinum neurotoxins (BoNTs; serotypes A-G) are metalloproteases, which cleave and inactivate cellular proteins essential for neurotransmitter release. In bacterial cultures, BoNTs are secreted as a complex of the neurotoxin and a group of neurotoxin associated proteins (NAPs). Under physiological condition (pH 7.4), this complex is believed to be dissociated to separate the neurotoxin from NAPs. BoNT consists of a 50 kDa light (L) chain (LC or catalytic domain) and a 100 kDa heavy (H) chain (or HC) linked through a disulfide bond and other non-covalent interactions. The cell intoxication involves three major steps; binding, membrane translocation and inhibition of neurotransmitter release. The last step of intoxication, endopeptidase activity, is very unique and specific that can be used for detection of the complex and isolated forms of the toxin. A fluorescent tag-labeled synthetic peptide (SNAPtide) derived from a segment of SNAP-25, an intracellular substrate of BoNT/A, is used to detect and assay the endopeptidase activity of BoNT/A. The detection of the signal is based on the change in the fluorescence energy transfer after selective cleavage of the peptide by the BoNT/A. In this report, we demonstrate that SNAPtide as a commonly used substrate widely differ in reaction with BoNT/A complex, BoNT/A, and BoNT/A light chain. These findings have implications for assays used in detection, and in screening potential inhibitors.

Asunto(s)

Toxinas Botulínicas Tipo A/metabolismo , Endopeptidasas/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Toxinas Botulínicas Tipo A/química , Dominio Catalítico , Clostridium botulinum/enzimología , Disulfuros/metabolismo , Endopeptidasas/química , Transferencia Resonante de Energía de Fluorescencia , Neurotoxinas/química , Neurotoxinas/metabolismo , Dominios Proteicos , Proteína 25 Asociada a Sinaptosomas/química

RESUMEN

Botulinum neurotoxins (BoNT) are among the most toxic known substances and currently there are no effective treatments for intraneuronal BoNT intoxication. Chicoric acid (ChA) was previously reported as a BoNT/A inhibitor that binds to the enzyme's α-exosite. Herein, we report the synthesis and structure-activity relationships (SARs) of a series of ChA derivatives, which revealed essential binding interactions between ChA and BoNT/A. Moreover, several ChA-based inhibitors with improved potency against the BoNT/A were discovered.

Asunto(s)

Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Ácidos Cafeicos/química , Inhibidores de Proteasas/química , Succinatos/química , Toxinas Botulínicas Tipo A/metabolismo , Ácidos Cafeicos/síntesis química , Ácidos Cafeicos/metabolismo , Clostridium botulinum/enzimología , Concentración 50 Inhibidora , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/metabolismo , Relación Estructura-Actividad , Succinatos/síntesis química , Succinatos/metabolismo

RESUMEN

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A-G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C-terminal heavy chain (HC ) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously-characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. HC /B1 and HC /B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its Hc .domain.

Asunto(s)

Toxinas Botulínicas Tipo A/metabolismo , Botulismo/microbiología , Clostridium botulinum/enzimología , Neurotoxinas/metabolismo , Toxinas Botulínicas Tipo A/química , Botulismo/metabolismo , Clostridium botulinum/química , Clostridium botulinum/genética , Gangliósidos/metabolismo , Humanos , Cinética , Neurotoxinas/química , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/metabolismo

RESUMEN

Botulinum neurotoxins are known to have seven serotypes (BoNT/A-G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells.

Asunto(s)

Toxinas Botulínicas/metabolismo , Botulismo/microbiología , Clostridium botulinum/enzimología , Neurotoxinas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Toxinas Botulínicas/química , Toxinas Botulínicas/genética , Toxinas Botulínicas/toxicidad , Botulismo/genética , Botulismo/metabolismo , Clostridium botulinum/genética , Humanos , Ratones , Modelos Moleculares , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/toxicidad , Proteínas R-SNARE/química , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Alineación de Secuencia , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo

RESUMEN

An esterase from Clostridium botulinum (Cbotu_EstA) previously reported to hydrolyze the biodegradable polyester poly(butylene adipate-co-terephthalate) was redesigned to improve the hydrolysis of synthetic polyesters. Increased activity was indeed observed for del71Cbotu_EstA variant, which performed activity on the widespread polyester polyethylene terephthalate, which was not able to be attacked by the wild-type enzyme Cbotu_EstA. Analysis of the 3D structure of the enzyme showed that removing 71 residues at the N-terminus of the enzyme exposed a hydrophobic patch on the surface and improved sorption of hydrophobic polyesters concomitantly facilitating the access of the polymer to the active site. These results show a new route for enhancing enzyme activity for hydrolysis and modification of polyesters.

Asunto(s)

Plásticos Biodegradables/química , Biodegradación Ambiental , Esterasas/química , Conformación Molecular , Dominio Catalítico/efectos de los fármacos , Clostridium botulinum/química , Clostridium botulinum/enzimología , Esterasas/metabolismo , Hidrólisis , Poliésteres/química , Tereftalatos Polietilenos/química

RESUMEN

Clostridium botulinum group III strains are able to produce cytotoxins, C2 toxin and C3 exotoxin, along with botulinum neurotoxin types C and D. C2 toxin and C3 exotoxin produced by this organism are the most important members of bacterial ADP-ribosyltransferase superfamily. Both toxins have distinct pathophysiological functions in the avian and mammalian hosts. The members of this superfamily transfer an ADP-ribose moiety of NAD+ to specific eukaryotic target proteins. The present review describes the structure, function and evolution aspects of these toxins with a special emphasis to the development of veterinary vaccines. C2 toxin is a binary toxin that consists of a catalytic subunit (C2I) and a translocation subunit (C2II). C2I component is structurally and functionally similar to the VIP2 and iota A toxin whereas C2II component shows a significant homology with the protective antigen from anthrax toxin and iota B. Unlike C2 toxin, C3 toxin is devoid of translocation/binding subunit. Extensive studies on their sequence-structure-function link spawn additional efforts to understand the catalytic mechanisms and target recognition. Structural and functional relationships with them are often determined by using evolutionary constraints as valuable biological measures. Enzyme-deficient mutants derived from these toxins have been used as drug/protein delivery systems in eukaryotic cells. Thus, current knowledge on their molecular diversity is a well-known perspective to design immunotoxin or subunit vaccine for C. botulinum infection.

Asunto(s)

Toxinas Botulínicas/química , Botulismo/veterinaria , Clostridium botulinum/patogenicidad , Evolución Molecular , Enfermedades de las Aves de Corral/microbiología , Aves de Corral/microbiología , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Vacunas Bacterianas/biosíntesis , Vacunas Bacterianas/inmunología , Toxinas Botulínicas/genética , Toxinas Botulínicas/metabolismo , Botulismo/microbiología , Botulismo/patología , Botulismo/prevención & control , Dominio Catalítico , Clostridium botulinum/clasificación , Clostridium botulinum/enzimología , Clostridium botulinum/genética , Expresión Génica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Filogenia , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/patología , Enfermedades de las Aves de Corral/prevención & control , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

Nature has provided a highly optimized toolbox in bacterial endotoxins with precise functions dictated by their clear structural division. Inspired by this streamlined design, a supramolecular approach capitalizing on the strong biomolecular (streptavidin (SA))-biotin interactions is reported herein to prepare two multipartite fusion constructs, which involves the generation 2.0 (D2) or generation 3.0 (D3) polyamidoamine-dendronized transporter proteins (dendronized streptavidin (D3SA) and dendronized human serum albumin (D2HSA)) non-covalently fused to the C3bot1 enzyme from Clostridium botulinum, a potent and specific Rho-inhibitor. The fusion constructs, D3SA-C3 and D2HSA-C3, represent the first examples of dendronized protein transporters that are fused to the C3 enzyme, and it is successfully demonstrated that the C3 Rho-inhibitor is delivered into the cytosol of mammalian cells as determined from the characteristic C3-mediated changes in cell morphology and confocal microscopy. The design circumvents the low uptake of the C3 enzyme by eukaryotic cells and holds great promise for reprogramming the properties of toxin enzymes using a supramolecular approach to broaden their therapeutic applications.

Asunto(s)

ADP Ribosa Transferasas/farmacología , Toxinas Botulínicas/farmacología , Dendrímeros/farmacología , Toxinas Biológicas/farmacología , ADP Ribosa Transferasas/química , Biotina/química , Toxinas Botulínicas/química , Línea Celular , Clostridium botulinum/enzimología , Citosol/efectos de los fármacos , Dendrímeros/química , Humanos , Macrófagos/efectos de los fármacos , Albúmina Sérica/química , Estreptavidina/química , Toxinas Biológicas/química

RESUMEN

Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.

Asunto(s)

Clostridium botulinum/enzimología , Esterasas/metabolismo , Poliésteres/metabolismo , Butiratos/metabolismo , Dominio Catalítico , Clostridium botulinum/química , Clostridium botulinum/metabolismo , Cristalografía por Rayos X , Esterasas/química , Hidrólisis , Modelos Moleculares , Nitrofenoles/metabolismo , Conformación Proteica , Especificidad por Sustrato , Zinc/metabolismo

RESUMEN

C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells.

Asunto(s)

ADP Ribosa Transferasas/farmacología , Toxinas Botulínicas/farmacología , Clostridium botulinum/química , Redes Reguladoras de Genes/efectos de los fármacos , Neuronas/efectos de los fármacos , Proteínas Nucleares/aislamiento & purificación , Proteoma/aislamiento & purificación , ADP Ribosa Transferasas/biosíntesis , ADP Ribosa Transferasas/genética , Animales , Toxinas Botulínicas/biosíntesis , Toxinas Botulínicas/genética , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Clostridium botulinum/enzimología , Clostridium botulinum/genética , Regulación de la Expresión Génica , Glucosa/metabolismo , Hipocampo/química , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Lisosomas/química , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Mitocondrias/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Anotación de Secuencia Molecular , Mutación , Neuronas/química , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biogénesis de Organelos , Cultivo Primario de Células , Biosíntesis de Proteínas/efectos de los fármacos , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Ribosomas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos

RESUMEN

Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.

Asunto(s)

Toxinas Botulínicas/análisis , Clostridium botulinum/enzimología , Técnicas Electroquímicas/métodos , Neurotoxinas/análisis , Técnicas Biosensibles/métodos , Toxinas Botulínicas/metabolismo , Botulismo/microbiología , Clostridium botulinum/aislamiento & purificación , Clostridium botulinum/metabolismo , Agua Potable/microbiología , Humanos , Límite de Detección , Neurotoxinas/metabolismo , Oligopéptidos/metabolismo , Oxidación-Reducción , Proteolisis

RESUMEN

The Clostridium botulinum C3 exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, suggesting a specific endocytotic pathway, which is not yet understood. In this study, we show that the C3 exoenzyme binds to cell surfaces and is internalized in a time-dependent manner. We show that the intermediate filament, vimentin, is involved in C3 uptake, as indicated by the inhibition of C3 internalization by acrylamide, a known vimentin disruption agent. Inhibition of C3 internalization was not observed by chemical inhibitors, like bafilomycin A, methyl-ß-cyclodextrin, nocodazole or latrunculin B. Furthermore, the internalization of C3 exoenzyme was markedly inhibited in dynasore-treated HT22 cells. Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae.

Asunto(s)

ADP Ribosa Transferasas/metabolismo , Toxinas Botulínicas/metabolismo , Dinaminas/metabolismo , Endocitosis , Vimentina/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Línea Celular , Clostridium botulinum/enzimología , Electroforesis en Gel de Poliacrilamida , Ratones , Microscopía Confocal , Unión Proteica , Receptores de Superficie Celular/metabolismo , Factores de Tiempo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA

RESUMEN

Botulinum neurotoxins (BoNTs) are among the most potent toxic bacterial proteins for humans, which make them potential agents for bioterrorism. Therefore, an ultrasensitive detection of BoNTs and their active states is in great need as field-deployable systems for anti-terrorism applications. We report the construction of a novel graphene oxide (GO)-peptide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of the BoNT serotype A light chain (BoNT-LcA) protease activity. A green fluorescence protein (GFP) modified SNAP-25 peptide substrate (SNAP-25-GFP) was optimally designed and synthesized with the centralized recognition/cleavage sites. This FRET platform was constructed by covalent immobilization of peptide substrate on GO with BSA passivation which have advantages of low non-specific adsorption and high stability in protein abundant solution. BoNT-LcA can specifically cleave SNAP-25-GFP substrate covalently immobilized on GO to release the fragment with GFP. Based on fluorescence signal recovery measurement, the target BoNT-LcA was detected sensitively and selectively with the linear detection range from 1fg/mL to 1pg/mL. The limit of detection (LOD) for BoNT-LcA is around 1fg/mL.

Asunto(s)

Toxinas Botulínicas Tipo A/análisis , Clostridium botulinum/enzimología , Transferencia Resonante de Energía de Fluorescencia/métodos , Grafito/química , Péptidos/química , Técnicas Biosensibles/métodos , Toxinas Botulínicas Tipo A/metabolismo , Botulismo/microbiología , Pruebas de Enzimas/métodos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Límite de Detección , Óxidos/química , Péptidos/metabolismo