RESUMO
Suramin was the first effective drug for the treatment of human African sleeping sickness. Structural analogues of the trypanocide have previously been shown to be potent inhibitors of several enzymes. Therefore, four suramin analogues lacking the methyl group on the intermediate rings and with different regiochemistry of the naphthalenetrisulphonic acid groups and the phenyl rings were tested to establish whether they exhibited improved antiproliferative activity against bloodstream forms of Trypanosomes brucei compared to the parent compound. The four analogues exhibited low trypanocidal activity and weak inhibition of the antitrypanosomal activity of suramin in competition experiments. This indicates that the strong trypanocidal activity of suramin is most likely due to the presence of methyl groups on its intermediate rings and to the specific regiochemistry of naphthalenetrisulphonic acid groups. These two structural features are also likely to be important for the inhibition mechanism of suramin because DNA distribution and nucleus/kinetoplast configuration analyses suggest that the analogues inhibit mitosis while suramin inhibits cytokinesis.
Assuntos
Suramina , Tripanossomicidas , Trypanosoma brucei brucei , Suramina/farmacologia , Suramina/química , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Relação Estrutura-Atividade , DNA de Protozoário/efeitos dos fármacos , DNA de Cinetoplasto/efeitos dos fármacos , Camundongos , Mitose/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologiaRESUMO
We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
Assuntos
Desenvolvimento de Medicamentos , Corantes Fluorescentes/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-AtividadeRESUMO
Detection of protein-binding analytes is important for many applications. Currently, various instrument-based techniques are used for detecting protein-binding analytes. However, such techniques have several limitations including high cost and time-consuming sample processing. In order to overcome these limitations, we developed a sensitive competition assay for the detection of protein-binding analytes using recombinant endospores as a sensing element. The method is based on the competition between the biotin, the model analyte, and a biotin-magnetic bead complex to bind the recombinant spores containing the biotin binding region of streptavidin. After magnetic attraction, the residual spores in the suspension are spread on plates to form colonies which are used to count the amount of the residual spores; the higher the residual ratio of spores, the more biotin in the samples. The linear range was from 150 zmol to 1.5 fmol and the limit of detection of the assay was 150 zmol. The assay proposed herein is sensitive and does not require any expensive equipment. It is suitable for qualitative or semi-quantitative analysis such as screening tests for the detection of toxic chemicals.
Assuntos
Biotina/análise , Esporos Bacterianos/química , Ensaio de Imunoadsorção Enzimática/métodos , Separação Imunomagnética , Limite de DetecçãoRESUMO
Evidence suggests that programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) targeted inhibitors act as an immune checkpoint blockade, indicating that these compounds may be useful in cancer immunotherapy by inhibiting the immune response between T-cells and tumors. Previous studies have shown that ginsenosides can regulate the expression of PD-1 and PD-L1 in target diseases; however, it remains unknown whether ginsenosides act as a blockade of PD-1/PD-L1 interactions. In this study, we used competitive ELISA to investigate 12 ginsenosides for their ability to block PD-1/PD-L1 interactions. In addition, we performed a protein-ligand docking simulation and examined the hydrophobic interactions and hydrogen bonds formed at the interfaces between the ginsenosides and PD-L1/PD-1. Eight out of the 12 ginsenosides studied showed inhibition of PD-1/PD-L1 interactions at 35% at the maximum concentration (1 µM). Among them, Rg3 and Compound K (C-K) demonstrated the highest inhibitory effects. Rg3 and C-K were further identified for their interaction efficacy with PD-1/PD-L1, which supported our results demonstrating the blocking activity of these compounds against PD-1/PD-L1 binding interactions. Collectively, our findings suggest that some ginsenosides, including Rg3 and C-K, inhibit PD-1/PD-L1 binding interactions. Therefore, these compounds may prove useful as part of an overall immuno-oncological strategy.
Assuntos
Antígeno B7-H1/metabolismo , Ginsenosídeos/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica/efeitos dos fármacos , Ginsenosídeos/química , Humanos , Inibidores de Checkpoint Imunológico/química , Inibidores de Checkpoint Imunológico/farmacologia , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
A common approach to measuring binding constants involves combining receptor and ligand and measuring the distribution of bound and free states after equilibration. For class I major histocompatibility (MHC-I) proteins, which bind short peptides for presentation to T cells, this approach is precluded by instability of peptide-free protein. Here we develop a method wherein a weakly-binding peptide covalently attached to the N-terminus of the MHC-I ß2m subunit is released from the peptide binding site after proteolytic cleavage of the linker. The resultant protein is able to bind added peptide. A direct binding assay and method for estimation of peptide binding constant (Kd) are described, in which fluorescence polarization is used to follow peptide binding. A competition binding assay and method for estimation of inhibitor binding constant (Ki) using the same principle also are also described. The method uses a cubic equation to relate observed binding to probe concentration, probe Kd, inhibitor concentration, and inhibitor Ki under general reaction conditions without assumptions relating to relative binding affinities or concentrations. We also delineate advantages of this approach compared to the Cheng-Prusoff and Munson-Rodbard approaches for estimation of Ki using competition binding data.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Proteólise , Microglobulina beta-2/metabolismo , Sequência de Aminoácidos , Peptídeos/química , Ligação ProteicaRESUMO
Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 ≤ induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments.
Assuntos
Proteínas Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Ligação Competitiva , Proteínas de Ciclo Celular , Quinase 9 Dependente de Ciclina/metabolismo , Células HeLa , Humanos , Cinética , Proteínas Mutantes/metabolismo , Mutação , Coativador 2 de Receptor Nuclear/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ligação Proteica , RatosRESUMO
A series of colchicine site binding tubulin inhibitors were synthesized by the modification of the combretastatin pharmacophore. The ring B was replaced by the pharmacologically relevant benzothiazole scaffolds, and the cis configuration of the olefinic bond was restricted by the incorporation of a triazole and tetrazole rings which is envisaged by the structural resemblance to a tubulin inhibitor like combretastatin (CA-4). These compounds were evaluated for their antiproliferative activity on selected cancer cell lines and an insight in the structure activity relationship was developed. The most potent compounds (9a and 9b) demonstrated an antiproliferative effect comparable to that of CA-4. Mitotic cell cycle arrest in G2/M phase revealed the disruption of microtubule dynamics that was confirmed by tubulin polymerization assays and immunocytochemistry studies at the cellular level. Western blot analysis revealed that these compounds accumulate more tubulin in the soluble fraction. The colchicine competitive binding assay and the molecular docking studies suggested that the binding of these mimics at the colchicine site of the tubulin is similar to that of CA-4. Moreover, the triggering of apoptotic cell death after mitotic arrest was investigated by studying their effect by Hoechst staining, Annexin-V-FITC assay, mitochondrial membrane potential, ROS generation and caspase-3 activation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Bibenzilas/farmacologia , Tetrazóis/farmacologia , Triazóis/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Benzotiazóis/síntese química , Benzotiazóis/química , Bibenzilas/síntese química , Bibenzilas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Polimerização/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/química , Triazóis/síntese química , Triazóis/químicaRESUMO
Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 µM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.
Assuntos
Calorimetria/métodos , Ligantes , Proteínas/química , Cloreto de Cálcio/química , Simulação por Computador , Ácido Edético/análogos & derivados , Ácido Edético/química , Ácido Egtázico/química , Inibidores Enzimáticos , Ligação Proteica , Proteínas/metabolismo , TermodinâmicaRESUMO
Saturated long chain-free fatty acids (FFAs), especially palmitate, have been implicated in apoptosis by inhibiting the activity of PKR (double-stranded RNA-dependent protein kinase). We recently found evidence that palmitate interacts directly with the kinase domain of PKR, subsequently inhibiting the autophosphorylation of PKR. To investigate the interactions of palmitate with PKR and its effects on PKR autophosphorylation, we performed extensive unbiased MD simulations combined with biochemical and biophysical experiments. The simulations predict multiple putative binding sites of palmitate on both the phosphorylated and unphosphorylated PKR with similar binding affinities. Ligand-protein interactions involving a large variety of different binding modes challenge the conventional view of highly specific, single binding sites. Key interactions of palmitate involve the αC-helix of PKR, especially near residue R307. Experimental mutation of R307 was found to affect palmitate binding and reduce its inhibitory effect. Based on this study a new allosteric mechanism is proposed where palmitate binding to the αC-helix prevents the inactive-to-active transition of PKR and subsequently reduces its ability to autophosphorylate.
Assuntos
Regulação da Expressão Gênica , Palmitatos/metabolismo , eIF-2 Quinase/química , eIF-2 Quinase/metabolismo , Sítios de Ligação , Western Blotting , Ativação Enzimática , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Fosforilação , Conformação Proteica , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , eIF-2 Quinase/genéticaRESUMO
Surely most chronobiologists believe circadian clocks are an adaptation of organisms that enhances fitness, but are we certain that this focus of our research effort really confers a fitness advantage? What is the evidence, and how do we evaluate it? What are the best criteria? These questions are the topic of this review. In addition, we will discuss selective pressures that might have led to the historical evolution of circadian systems while considering the intriguing question of whether the ongoing climate change is modulating these selective pressures so that the clock is still evolving.
Assuntos
Relógios Circadianos , Relógios Circadianos/genética , Ritmo CircadianoRESUMO
Here we present LUCOS (Luminescent Competition Sensor), a modular and broadly applicable bioluminescent diagnostic platform enabling the detection of both small molecules and protein biomarkers. The construction of LUCOS sensors entails the covalent and site-specific coupling of a bioluminescent sensor component to an analyte-specific antibody via protein G-mediated photoconjugation. Target detection is accomplished through intramolecular competition with a tethered analyte competitor for antibody binding. We established two variants of LUCOS: an inherent ratiometric LUCOSR variant and an intensiometric LUCOSI version, which can be used for ratiometric detection upon the addition of a split calibrator luciferase. To demonstrate the versatility of the LUCOS platform, sensors were developed for the detection of the small molecule cortisol and the protein biomarker NT-proBNP. Sensors for both targets displayed analyte-dependent changes in the emission ratio and enabled detection in the micromolar concentration range (KD,app = 16-92 µM). Furthermore, we showed that the response range of the LUCOS sensor can be adjusted by attenuating the affinity of the tethered NT-proBNP competitor, which enabled detection in the nanomolar concentration range (KD,app = 317 ± 26 nM). Overall, the LUCOS platform offers a highly versatile and easy method to convert commercially available monoclonal antibodies into bioluminescent biosensors that provide a homogeneous alternative for the competitive immunoassay.
Assuntos
Anticorpos Monoclonais , Luciferases , Imunoensaio/métodosRESUMO
RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5' conserved sequence element that disrupt sequence but not structure did not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) did cause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent, primary sequence determinants in this loop play an important role in CHIKV biology. Our novel findings illustrate the utility of the U-CAN-seq competition assay.
Assuntos
Vírus Chikungunya , Mutação , Sequenciamento por Nanoporos , Sequenciamento por Nanoporos/métodos , Vírus Chikungunya/genética , Vírus Chikungunya/classificação , Humanos , Genótipo , Aptidão Genética , RNA Viral/genética , Animais , Vírus de RNA/genética , Vírus de RNA/classificação , Febre de Chikungunya/virologiaRESUMO
Multiple Salmonella enterica serovars and strains have been reported to be able to persist inside the foliar tissue of lettuce (Lactuca sativa L.), potentially resisting washing steps and reaching the consumer. Intraspecies variation of the bacterial pathogen and of the plant host can both significantly affect the outcome of foliar colonization. However, current understanding of the mechanisms underlying this phenomenon is still very limited. In this study, we evaluated the foliar fitness of 14 genetically barcoded S. enterica isolates from 10 different serovars, collected from plant and animal sources. The S. enterica isolates were vacuum-infiltrated individually or in pools into the leaves of three- to four-week-old lettuce plants. To estimate the survival capacity of individual isolates, we enumerated the bacterial populations at 0- and 10- days post-inoculation (DPI) and calculated their net growth. The competition of isolates in the lettuce apoplast was assessed through the determination of the relative abundance change of barcode counts of each isolate within pools during the 10 DPI experimental period. Isolates exhibiting varying apoplast fitness phenotypes were used to evaluate their capacity to grow in metabolites extracted from the lettuce apoplast and to elicit the reactive oxygen species burst immune response. Our study revealed that strains of S. enterica can substantially differ in their ability to survive and compete in a co-inhabited lettuce leaf apoplast. The differential foliar fitness observed among these S. enterica isolates might be explained, in part, by their ability to utilize nutrients available in the apoplast and to evade plant immune responses in this niche.
RESUMO
Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.
Assuntos
Proteínas de Homeodomínio , Proteínas do Tecido Nervoso , Humanos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Mutação , Células HEK293 , Transtorno do Espectro Autista , Cardiopatias , Fácies , Transtornos do NeurodesenvolvimentoRESUMO
Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC). Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target. Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.
Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.
RESUMO
Understanding how ecological communities assemble is an urgent research priority. In this study, we used a community ecology approach to examine how ecological and evolutionary processes shape biodiversity patterns of plant pathogenic fungi, Fusarium graminearum and F. asiaticum. High-throughput screening revealed that the isolates had a wide range of phenotypic variation in stress tolerance traits. Net Relatedness Index (NRI) and Nearest Taxon Index (NTI) values were computed based on stress-tolerant distance matrices. Certain local regions exhibited positive values of NRI and NTI, indicating phenotypic clustering within the fungal communities. Competition assays of the pooled strains were conducted to investigate the cause of clustering. During stress conditions and wheat colonization, only a few strains dominated the fungal communities, resulting in reduced diversity. Overall, our findings support the modern coexistence theory that abiotic stress and competition lead to phenotypic similarities among coexisting organisms by excluding large, low-competitive clades. We suggest that agricultural environments and competition for host infection lead to locally clustered communities of plant pathogenic fungi in the field.
RESUMO
Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The KD value observed is in accordance with the IC50 determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.
Assuntos
Vírus da Dengue , Vírus da Dengue/metabolismo , Peptídeo Hidrolases/metabolismo , Ligantes , Transferência Ressonante de Energia de Fluorescência , Proteínas não Estruturais Virais/química , Antivirais/químicaRESUMO
Isothermal titration calorimetry (ITC) is the gold standard for providing quantitative and thermodynamic understanding of the interaction mechanisms between core autophagy machinery, autophagy receptors, and ATG8. Here, we used two model peptides and Arabidopsis thaliana ATG8A to characterize ATG8-peptide interactions. We employed ITC using three different methods (direct ligand titration, displacement, and competition assays) to characterize, directly and indirectly, the interaction of the peptides with ATG8. We then analyzed the ITC data by global and statistical methods and discussed advantages, drawbacks, and negative controls for each approach. We finally provide a thorough description of all the steps, including data analysis and presentation, preparation of recombinant ATG8A from E. coli, and troubleshooting notes for technical problems that can be encountered. Although we used ATG8-peptide interactions here, these assays can be applied to any other one-to-one protein-protein and ligand-protein interactions and competitive binders.
Assuntos
Escherichia coli , Peptídeos , Ligantes , Ligação Proteica , Escherichia coli/genética , Calorimetria/métodosRESUMO
Competition assays are a simple phenotyping strategy that confront two bacterial strains to evaluate their relative fitness. Because they are more accurate than single-strain growth assays, competition assays can be used to highlight slight differences that would not otherwise be detectable. In the frame of host-pathogens interactions, they can be very useful to study the contribution of individual bacterial genes to bacterial fitness and lead to the identification of new adaptive traits. Here, we describe how to perform such competition assays by taking the example of the model phytopathogenic bacterium Xanthomonas campestris pv. campestris during infection of the mesophyll of its cauliflower host. This phenotypic assay is based on the use of a Competitive Index (CI) that compares the relative abundance of co-inoculated strains before and after inoculation. Since multiplication is a direct proxy for bacterial fitness, the evolution of the ratio between both strains in the mixed population is a direct way to assess differences in fitness in a given environment. In this protocol, we exploit the blue staining of GUS-expressing bacteria to count blue vs. white colonies on plates and estimate the competitiveness of the strains of interest in plant mesophyll.
RESUMO
Candida albicans populations present in the mammalian gastrointestinal tract are a major source of candidemia and subsequent severe invasive candidiasis in those individuals with acquired or congenital immune defects. Understanding the mechanisms used by this fungus to colonize this niche is, therefore, of primary importance to develop new therapeutic options that could lead to control its proliferation in the host. The recent popularization of models of commensalism in mice combined with the already powerful tools in C. albicans genetics allows to analyze the role of specific genes during colonization. Fitness can be analyzed for a specific C. albicans strain (test strain) by comparing its growth in vivo with an otherwise isogenic control strain via the analysis of the luminal content of the mouse gastrointestinal tract using flow cytometry, qPCR, or viable fungal cell counting. While all these procedures have limitations, they can be used to estimate the degree of adaptation of the test strain to the mammalian tract by determining its relative abundance with an internal control strain. By using specific genetically engineered C. albicans and mouse strains, antibiotic regimes, or even germ-free mice, this methodology allows to determine the role of the host immunological status, the bacterial microbiota, or individual fungal features (e.g., dimorphism) in the process of colonization of C. albicans of the mammalian gut.