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Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA1 induces auto-phosphorylation of MenT1 by repositioning the MenT1 phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA3 is similarly able to induce auto-phosphorylation of cognate toxin MenT3. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.
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Dominio Catalítico , Mycobacterium tuberculosis , Nucleotidiltransferasas , Fosforilación , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Modelos Moleculares , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Cristalografía por Rayos XRESUMEN
A class of chemical with a potentially important perceived future contribution to the net zero carbon goal (as "green" solvents) is the methylimidazolium ionic liquids (MILs). These solvents are used in industrial processes such as biofuel production yet little is known about their environmental stability or toxicity in man although one MIL - 1-octyl-3-methylimidazolium (M8OI) - has been shown to activate the human estrogen receptor alpha (ERα). The stabilities of the chloride unsubstituted methylimidazolium (MI) and MILs possessing increasing alkyl chain lengths (2C, 1-ethyl-3-methylimidazolium (EMI); 4C, 1-butyl-3-methylimidazolium (BMI); 6C; 1-hexyl-3-methylimidazolium (HMI), 8C, M8OI; 10C, 1-decyl-3-methylimidazolium (DMI)) were examined in river water and a human liver model system. The MILs were also screened for their abilities to activate the human ERα in vitro and induce uterine growth in pre-pubertal rats in vivo. Short chain MILs (EMI, BMI and HMI) underwent negligible metabolism and mineralisation in river water; were not metabolised in a model of human liver metabolism; activated the human ERα in vitro and were estrogenic in vivo in rats. A structure-based computational approach predicted short chain MIL binding to both the estrogen binding site and an additional site on the human estrogen receptor alpha. Longer chain MILs (M8OI and DMI) were metabolised in river water and partially mineralised. Based on structure-activity considerations, some of these environmentally-derived metabolites may however, remain a hazard to the population. MILs therefore have the potential to become forever chemicals with adverse effects to both man, other animals and the environment in general.
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Disruptores Endocrinos , Receptor alfa de Estrógeno , Imidazoles , Líquidos Iónicos , Líquidos Iónicos/toxicidad , Líquidos Iónicos/química , Imidazoles/toxicidad , Imidazoles/química , Disruptores Endocrinos/toxicidad , Disruptores Endocrinos/química , Humanos , Receptor alfa de Estrógeno/metabolismo , Animales , Ratas , FemeninoRESUMEN
INTRODUCTION: Complement-based drug discovery is undergoing a renaissance, empowered by new advances in structural biology, complement biology and drug development. Certain components of the complement pathway, particularly C1q and C3, have been extensively studied in the context of neurodegenerative disease, and established as key therapeutic targets. C5 also has huge therapeutic potential in this arena, with its druggability clearly demonstrated by the success of C5-inhibitor eculizumab. AREAS COVERED: We will discuss the evidence supporting C5 as a target in neurodegenerative disease, along with the current progress in developing different classes of C5 inhibitors and the gaps in knowledge that will help progress in the field. EXPERT OPINION: Validation of C5 as a therapeutic target for neurodegenerative disease would represent a major step forward for complement therapeutics research and has the potential to furnish disease-modifying drugs for millions of patients suffering worldwide. Key hurdles that need to be overcome for this to be achieved are understanding how C5a and C5b should be targeted to bring therapeutic benefit and demonstrating the ability to target C5 without creating vulnerability to infection in patients. This requires greater biological elucidation of its precise role in disease pathogenesis, supported by better chemical/biological tools.
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Complemento C5 , Enfermedades Neurodegenerativas , Humanos , Complemento C5/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Activación de Complemento , Complemento C5aRESUMEN
The androgen receptor (AR) is an important drug target in prostate cancer and a driver of castration-resistant prostate cancer (CRPC). A significant challenge in designing effective drugs lies in targeting constitutively active AR variants and, most importantly, nearly all AR variants lacking the ligand-binding domain (LBD). Recent findings show that an AR's constitutive activity may occur in the presence of somatic DNA mutations within non-coding regions, but the role of these mutations remains elusive. The discovery of new drugs targeting CRPC is hampered by the limited molecular understanding of how AR binds mutated DNA sequences, frequently observed in prostate cancer, and how mutations within the protein and DNA regulate AR-DNA interactions. Using atomistic molecular dynamics (MD) simulations and quantum mechanical calculations, we focused our efforts on (i) rationalising the role of several activating DBD mutations linked to prostate cancer, and (ii) DBD interactions in the presence of abasic DNA lesions, which frequently occur in CRPC. Our results elucidate the role of mutations within DBD through their modulation of the intrinsic dynamics of the DBD-DNA ternary complex. Furthermore, our results indicate that the DNA apurinic lesions occurring in the androgen-responsive element (ARE) enhance direct AR-DNA interactions and stabilise the DBD homodimerisation interface. Moreover, our results strongly suggest that those abasic lesions may form reversible covalent crosslinks between DNA and lysine residues of an AR via a Schiff base. In addition to providing an atomistic model explaining how protein mutations within the AR DNA-binding domain affect AR dimerisation and AR-DNA interactions, our findings provide insight into how somatic mutations occurring in DNA non-coding regions may activate ARs. These mutations are frequently observed in prostate cancer and may contribute to disease progression by enhancing direct AR-DNA interactions.
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Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Andrógenos/metabolismo , Dominios Proteicos , ADN/genéticaRESUMEN
Mitophagy, the elimination of mitochondria via the autophagy-lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin-dependent mitophagy. We identified redox-sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.
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Mitofagia , Proteínas Nucleares , Proteínas Quinasas , Humanos , Autofagia , Células HeLa , Mitofagia/fisiología , Oxidación-Reducción , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
By harnessing the chirality of the DNA double helix, chemists have been able to obtain new, reliable, selective, and environmentally friendly biohybrid catalytic systems with tailor-made functions. Nonetheless, despite all the advances made throughout the years in the field of DNA-based asymmetric catalysis, many challenges still remain to be faced, in particular when it comes to designing a "universal" catalyst with broad reactivity and unprecedented selectivity. Rational design and rounds of selection have allowed us to approach this goal. We report here the development of a DNA/RNA hybrid catalytic system featuring a covalently attached bipyridine ligand, which exhibits unmatched levels of selectivity throughout the current DNA toolbox and opens new avenues in asymmetric catalysis.
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NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.
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Alquinos/farmacología , Cisteína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Alquinos/síntesis química , Alquinos/química , Cisteína/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Quinasa de Factor Nuclear kappa BRESUMEN
Cholinergic α7 nicotinic receptors encoded by the CHRNA7 gene are ligand-gated ion channels directly related to memory and immunomodulation. Exons 5-7 in CHRNA7 can be duplicated and fused to exons A-E of FAR7a, resulting in a hybrid gene known as CHRFAM7A, unique to humans. Its product, denoted herein as Dupα7, is a truncated subunit where the N-terminal 146 residues of the ligand binding domain of the α7 receptor have been replaced by 27 residues from FAM7. Dupα7 negatively affects the functioning of α7 receptors associated with neurological disorders, including Alzheimer's diseases and schizophrenia. However, the stoichiometry for the α7 nicotinic receptor containing dupα7 monomers remains unknown. In this work, we developed computational models of all possible combinations of wild-type α7 and dupα7 pentamers and evaluated their stability via atomistic molecular dynamics and coarse-grain simulations. We assessed the effect of dupα7 subunits on the Ca2+ conductance using free energy calculations. We showed that receptors comprising of four or more dupα7 subunits are not stable enough to constitute a functional ion channel. We also showed that models with dupα7/α7 interfaces are more stable and are less detrimental for the ion conductance in comparison to dupα7/dupα7 interfaces. Based on these models, we used protein-protein docking to evaluate how such interfaces would interact with an antagonist, α-bungarotoxin, and amyloid Aß42. Our findings show that the optimal stoichiometry of dupα7/α7 functional pentamers should be no more than three dupα7 monomers, in favour of a dupα7/α7 interface in comparison to a homodimer dupα7/dupα7 interface. We also showed that receptors bearing dupα7 subunits are less sensitive to Aß42 effects, which may shed light on the translational gap reported for strategies focused on nicotinic receptors in 'Alzheimer's disease research.
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Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Exones/genética , Duplicación de Gen/genética , Humanos , Ligandos , RatonesRESUMEN
Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing a pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between a leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor's island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of the PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel "druggable" binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.
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Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/química , Arabidopsis/química , Simulación de Dinámica Molecular , Reguladores del Crecimiento de las Plantas/química , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ligandos , Reguladores del Crecimiento de las Plantas/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Receptores de Superficie Celular/metabolismoRESUMEN
Monoclonal antibodies (mAbs) constitute a rapidly growing biopharmaceutical sector. However, their growth is impeded by high failure rates originating from failed clinical trials and developability issues in process development. There is, therefore, a growing need for better in silico tools to aid in risk assessment of mAb candidates to promote early-stage screening of potentially problematic mAb candidates. In this study, a quantitative structure-activity relationship (QSAR) modelling workflow was designed for the prediction of hydrophobic interaction chromatography (HIC) retention times of mAbs. Three novel descriptor sets derived from primary sequence, homology modelling, and atomistic molecular dynamics (MD) simulations were developed and assessed to determine the necessary level of structural resolution needed to accurately capture the relationship between mAb structures and HIC retention times. The results showed that descriptors derived from 3D structures obtained after MD simulations were the most suitable for HIC retention time prediction with a R2 = 0.63 in an external test set. It was found that when using homology modelling, the resulting 3D structures became biased towards the used structural template. Performing an MD simulation therefore proved to be a necessary post-processing step for the mAb structures in order to relax the structures and allow them to attain a more natural conformation. Based on the results, the proposed workflow in this paper could therefore potentially contribute to aid in risk assessment of mAb candidates in early development.
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Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Fragmentos Fab de Inmunoglobulinas/análisis , Fragmentos Fab de Inmunoglobulinas/química , Simulación de Dinámica Molecular , Anticuerpos Monoclonales/aislamiento & purificación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Modelos Químicos , Relación Estructura-Actividad CuantitativaRESUMEN
In the elderly population, pathological inflammation has been associated with ageing-associated diseases. The term 'inflammageing', which was used for the first time by Franceschi and co-workers in 2000, is associated with the chronic, low-grade, subclinical inflammatory processes coupled to biological ageing. The source of these inflammatory processes is debated. The senescence-associated secretory phenotype (SASP) has been proposed as the main origin of inflammageing. The SASP is characterised by the release of inflammatory cytokines, elevated activation of the NLRP3 inflammasome, altered regulation of acetylcholine (ACh) nicotinic receptors, and abnormal NAD+ metabolism. Therefore, SASP may be 'druggable' by small molecule therapeutics targeting those emerging molecular targets. It has been shown that inflammageing is a hallmark of various cardiovascular diseases, including atherosclerosis, hypertension, and adverse cardiac remodelling. Therefore, the pathomechanism involving SASP activation via the NLRP3 inflammasome; modulation of NLRP3 via α7 nicotinic ACh receptors; and modulation by senolytics targeting other proteins have gained a lot of interest within cardiovascular research and drug development communities. In this review, which offers a unique view from both clinical and preclinical target-based drug discovery perspectives, we have focused on cardiovascular inflammageing and its molecular mechanisms. We have outlined the mechanistic links between inflammageing, SASP, interleukin (IL)-1ß, NLRP3 inflammasome, nicotinic ACh receptors, and molecular targets of senolytic drugs in the context of cardiovascular diseases. We have addressed the 'druggability' of NLRP3 and nicotinic α7 receptors by small molecules, as these proteins represent novel and exciting targets for therapeutic interventions targeting inflammageing in the cardiovascular system and beyond.
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Envejecimiento , Inflamación/patología , Terapia Molecular Dirigida , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Intrinsically disordered proteins (IDPs) are molecules without a fixed tertiary structure, exerting crucial roles in cellular signalling, growth and molecular recognition events. Due to their high plasticity, IDPs are very challenging in experimental and computational structural studies. To provide detailed atomic insight in IDPs' dynamics governing their functional mechanisms, all-atom molecular dynamics (MD) simulations are widely employed. However, the current generalist force fields and solvent models are unable to generate satisfactory ensembles for IDPs when compared to existing experimental data. In this work, we present a new solvation model, denoted as the Charge-Augmented Three-Point Water Model for Intrinsically Disordered Proteins (CAIPi3P). CAIPi3P has been generated by performing a systematic scan of atomic partial charges assigned to the widely popular molecular scaffold of the three-point TIP3P water model. We found that explicit solvent MD simulations employing CAIPi3P solvation considerably improved the small-angle X-ray scattering (SAXS) scattering profiles for three different IDPs. Not surprisingly, this improvement was further enhanced by using CAIPi3P water in combination with the protein force field parametrized for IDPs. We also demonstrated the applicability of CAIPi3P to molecular systems containing structured as well as intrinsically disordered regions/domains. Our results highlight the crucial importance of solvent effects for generating molecular ensembles of IDPs which reproduce the experimental data available. Hence, we conclude that our newly developed CAIPi3P solvation model is a valuable tool for molecular simulations of intrinsically disordered proteins and assessing their molecular dynamics.
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Proteínas Intrínsecamente Desordenadas/química , Solventes/química , Agua/química , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) act as oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here we show that the key determinant of Grx function is a distinct loop structure adjacent to the active site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its function from oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-type loop abolished FeS transferase activity and activated the oxidative half reaction of the oxidoreductase. The reductive half-reaction, requiring the interaction with a second GSH molecule, was enabled by switching additional residues in the active site. We explain how subtle structural differences, mostly depending on the structure of one particular loop, act in concert to determine Grx function.
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Glutarredoxinas/metabolismo , Animales , Dominio Catalítico , Glutarredoxinas/química , Humanos , Proteínas Hierro-Azufre/química , Oxidación-Reducción , Unión Proteica , Estructura Secundaria de Proteína , Transducción de Señal/fisiología , Especificidad por SustratoRESUMEN
Cosolvent Molecular Dynamics (MD) simulations are increasingly popular techniques developed for prediction and characterization of allosteric and cryptic binding sites, which can be rendered "druggable" by small molecule ligands. Despite their conceptual simplicity and effectiveness, the analysis of cosolvent MD trajectories relies on pocket volume data, which requires a high level of manual investigation and may introduce a bias. In this work, we present CAT (Cosolvent Analysis Toolkit): an open-source, freely accessible analytical tool, suitable for automated analysis of cosolvent MD trajectories. CAT is compatible with commonly used molecular graphics software packages such as UCSF Chimera and VMD. Using a novel hybrid empirical force field scoring function, CAT accurately ranks the dynamic interactions between the macromolecular target and cosolvent molecules. To benchmark, CAT was used for three validated protein targets with allosteric and orthosteric binding sites, using five chemically distinct cosolvent molecules. For all systems, CAT has accurately identified all known sites. CAT can thus assist in computational studies aiming at identification of protein "hotspots" in a wide range of systems. As an easy-to-use computational tool, we expect that CAT will contribute to an increase in the size of the potentially 'druggable' human proteome.
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Ligandos , Sustancias Macromoleculares/química , Simulación de Dinámica Molecular , Proteoma , Programas Informáticos , Solventes/química , 2-Propanol/química , Acetamidas/química , Acetanilidas/química , Algoritmos , Benceno/química , Sitios de Unión , Análisis por Conglomerados , Humanos , Imidazoles/química , Dominios Proteicos , Proteínas/química , Receptores Androgénicos/químicaRESUMEN
Signal transducer activator of transcription 3 (STAT3) is among the most investigated oncogenic transcription factors, as it is highly associated with cancer initiation, progression, metastasis, chemoresistance, and immune evasion. Evidences from both preclinical and clinical studies have demonstrated that STAT3 plays a critical role in several malignancies associated with poor prognosis such as glioblastoma and triple-negative breast cancer, and STAT3 inhibitors have shown efficacy in inhibiting cancer growth and metastasis. Constitutive activation of STAT3 by mutations occurs frequently in tumor cells and directly contributes to many malignant phenotypes. Unfortunately, detailed structural biology studies on STAT3 as well as target-based drug discovery efforts have been hampered by difficulties in the expression and purification of the full-length STAT3 and a lack of ligand-bound crystal structures. Considering these, molecular modeling and simulations offer an attractive strategy for the assessment of the "druggability" of STAT3 dimers and allow investigations of reported activating and inhibiting STAT3 mutants at the atomistic level of detail. In the present study, we focused on the effects exerted by reported STAT3 mutations on the protein structure, dynamics, DNA-binding, and dimerization, thus linking structure, dynamics, energetics, and the biological function. By employing atomistic molecular dynamics and umbrella-sampling simulations to a series of human STAT3 dimers, which comprised wild-type protein and four mutations, we explained the modulation of STAT3 activity by these mutations. Counter-intuitively, our results show that the D570K inhibitory mutation exerts its effect by enhancing rather than weakening STAT3-DNA interactions, which interfere with the DNA release by the protein dimer and thus inhibit STAT3 function as a transcription factor. We mapped the binding site and characterized the binding mode of a clinical candidate napabucasin/BBI-608 at STAT3, which resembles the effect of a D570K mutation. Our results contribute to understanding the activation/inhibition mechanism of STAT3, to explain the molecular mechanism of STAT3 inhibition by BBI-608. Alongside the characterization of the BBI-608 binding mode, we also discovered a novel binding site amenable to bind small-molecule ligands, which may pave the way to design novel STAT3 inhibitors and to suggest new strategies for pharmacological interventions to combat cancers associated with poor prognosis.
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Per-Arnt-Sim (PAS) domains are key regions that occur in different regulatory proteins from all kingdoms of life. PAS domains show a remarkably conserved structural scaffold, despite a highly variable primary sequence. In this study we have attempted to address some of the gaps in knowledge regarding the druggability of PAS-A domains, differences in structure and dynamics within the PAS domain family and how this affects the druggability potential, as well as give insight into the druggability of steroid receptor coactivators and putative binding modes of the NCOA1. Investigations were performed through a range of computational methods including molecular docking studies, atomistic molecular dynamics simulations, and hotspot mapping. Atomistic molecular dynamics simulations show that the function of the AhR PAS-B domain is regulated by the dynamics of the highly conserved tyrosine Y322 residue, which acts as a "gatekeeper" controlling the access to the binding cavity and finely tuning the binding affinity. Furthermore, the transition between the partially unfolded and helical conformation of the loop1 segment within PAS-B domains was shown to be essential for the generation of "druggable" sites, especially for the NCOA1 PAS-B domain. Finally, our simulations indicated the undruggability of PAS-A domains, caused by the inherent characteristics of their putative binding sites. In conclusion, this work emphasises the role of intrinsic dynamics in tuning the druggability of PAS-B domains and shows that PAS-B domains of steroid receptor coactivators, such as NCOA1, can be targeted by small molecule ligands, which highlights the potential of developing new therapeutics designed to target these coactivators using structure-based approaches.
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The on-column selective conversion of guanosine to thioguanosine (tG) yields modified oligomers that exhibit destabilisation over the fully complementary duplex. Restoration to a stabilised duplex is induced through thio-directed Cd2+ coordination; a route for healing DNA damage. Short oligomers are G-specifically thiolated through a modified on-column protocol without the need for costly thioguanosine phosphoramidites. Addition of Cd2+ ions to a duplex containing a highly disrupted tG central mismatch sequence, 3'-A6 tG4 T6 -5', suggests a (tG)8 Cd2 central coordination regime, resulting in increased base stacking and duplex stability. Equilibrium molecular dynamic calculations support the hypothesis of metal-induced healing of the thiolated duplex. The 2â nm displacement of the central tG mismatched region is dramatically reduced after the addition of a chemical stimuli, Cd2+ ions, returning to a minimized fluctuational state comparable to the unmodified fully complementary oligomer.
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Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress.
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Aldehído Deshidrogenasa/genética , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Estrés Oxidativo/genética , Staphylococcus aureus/metabolismo , Aldehído Deshidrogenasa/química , Antibacterianos/química , Dominio Catalítico , Cisteína/biosíntesis , Cisteína/genética , Glucosamina/biosíntesis , Glucosamina/genética , Peróxido de Hidrógeno/química , Ácido Hipocloroso/toxicidad , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Proteína S/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidadRESUMEN
Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.
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Autofagia , Proteostasis , Especies Reactivas de Oxígeno/metabolismo , Proteína Sequestosoma-1/metabolismo , Secuencia de Aminoácidos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Ratones Noqueados , Oxidantes/farmacología , Oxidación-Reducción , Homología de Secuencia de Aminoácido , Proteína Sequestosoma-1/genéticaRESUMEN
Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures. Finally, comparative molecular docking combined with molecular dynamics and free energy of binding calculations were carried out to search for ligands showing selective affinity to T. spiralis TS. Despite an overall strong similarity in structure and dynamics of nematode vs mammalian TSs, a pool of ligands demonstrating predictively a strong and selective binding to TsTS has been delimited. These compounds, the E63 family, locate in the dimerization interface of TsTS where they exert species-specific interactions with certain non-conserved residues, including hydrogen bonds with Thr174 and hydrophobic contacts with Phe192, Cys191 and Tyr152. The E63 family of ligands opens the possibility of future development of selective inhibitors of TsTS and effective agents against trichinellosis.