RESUMO
The characterization of the statistical ensemble of conformations of intrinsically disordered regions (IDRs) is a great challenge both from experimental and computational points of view. In this respect, a number of protocols have been developed using molecular dynamics (MD) simulations to sample the huge conformational space of the molecule. In this work, we consider one of the best methods available, replica exchange solute tempering (REST), as a reference to compare the results obtained using this method with the results obtained using other methods, in terms of experimentally measurable quantities. Along with the methods assessed, we propose here a novel protocol called probabilistic MD chain growth (PMD-CG), which combines the flexible-meccano and hierarchical chain growth methods with the statistical data obtained from tripeptide MD trajectories as the starting point. The system chosen for testing is a 20-residue region from the C-terminal domain of the p53 tumor suppressor protein (p53-CTD). Our results show that PMD-CG provides an ensemble of conformations extremely quickly, after suitable computation of the conformational pool for all peptide triplets of the IDR sequence. The measurable quantities computed on the ensemble of conformations agree well with those based on the REST conformational ensemble.
RESUMO
The nature of the nanoparticle-protein corona is emerging as a key aspect in determining the impact of nanomaterials on proteins and in general on the biological response. We previously demonstrated that citrate-stabilized gold nanoparticles (Cit-AuNPs) interact with ß2-microglobulin (ß2m) preserving the protein native structure. Moreover, Cit-AuNPs are able to hinder in vitro fibrillogenesis of a ß2m pathologic variant, namely D76N, by reducing the oligomeric association of the protein in solution. Here, we clarify the characteristics of the interaction between ß2m and Cit-AuNPs by means of different techniques, i.e. surface enhanced Raman spectroscopy, NMR and quartz crystal microbalance with dissipation monitoring. All the results obtained clearly show that by simply changing the ionic strength of the medium it is possible to switch from a labile and transient nature of the protein-NP adduct featuring the so-called soft corona, to a more "hard" interaction with a layer of proteins having a longer residence time on the NP surface. This confirms that the interaction between ß2m and Cit-AuNPs is dominated by electrostatic forces which can be tuned by modifying the ionic strength.
Assuntos
Nanopartículas Metálicas/química , Coroa de Proteína/química , Microglobulina beta-2/química , Citratos/química , Ouro/química , Mutação , Concentração Osmolar , Eletricidade Estática , Microglobulina beta-2/genéticaRESUMO
MOTIVATION: Implicit solvent models play an important role in describing the thermodynamics and the dynamics of biomolecular systems. Key to an efficient use of these models is the computation of generalized Born (GB) radii, which is accomplished by algorithms based on the electrostatics of inhomogeneous dielectric media. The speed and accuracy of such computations are still an issue especially for their intensive use in classical molecular dynamics. Here, we propose an alternative approach that encodes the physics of the phenomena and the chemical structure of the molecules in model parameters which are learned from examples. RESULTS: GB radii have been computed using (i) a linear model and (ii) a neural network. The input is the element, the histogram of counts of neighbouring atoms, divided by atom element, within 16 Å. Linear models are ca. 8 times faster than the most widely used reference method and the accuracy is higher with correlation coefficient with the inverse of 'perfect' GB radii of 0.94 versus 0.80 of the reference method. Neural networks further improve the accuracy of the predictions with correlation coefficient with 'perfect' GB radii of 0.97 and ca. 20% smaller root mean square error. AVAILABILITY AND IMPLEMENTATION: We provide a C program implementing the computation using the linear model, including the coefficients appropriate for the set of Bondi radii, as Supplementary Material. We also provide a Python implementation of the neural network model with parameter and example files in the Supplementary Material as well. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Redes Neurais de Computação , Modelos Lineares , Solventes , Eletricidade Estática , TermodinâmicaRESUMO
SPG6 accounts for 1% of autosomal dominant Hereditary Spastic Paraplegia (HSP) and is caused by pathogenic variants in NIPA1, which encodes a magnesium transporter located in plasma membrane and early endosomes, implicated in neuronal development and maintenance. Here we report a 39-year-old woman affected by progressive gait disturbance associated to absence seizures episodes within childhood. Clinical exome sequencing identified a likely pathogenic de novo heterozygous variant in NIPA1 (NM_144599.5 c.249 C > G; p.Asn83Lys). Molecular modelling was performed to evaluate putative functional consequence of the NIPA1 protein. Indeed, the Asn83Lys modification is predicted to induce a significant perturbation of the protein structure, altering signal transduction or small-molecule transport by modulating the length of the second transmembrane domain. This is the first study reporting a SPG6-affected patient harbouring the NIPA1 p.Asn83Lys mutation.
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Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/genética , Adulto , Alelos , Substituição de Aminoácidos , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , FenótipoRESUMO
BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.
Assuntos
Anticorpos Monoclonais/química , Cadeias Pesadas de Imunoglobulinas/química , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Anticorpos de Domínio Único/química , Microglobulina beta-2/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Elementos Estruturais de Proteínas , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Microglobulina beta-2/imunologiaRESUMO
The permeability transition pore (PTP) is a Ca2+-dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (F)-ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP-dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch-clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F-ATP synthase.
Assuntos
Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Bovinos , Linhagem Celular , Permeabilidade da Membrana Celular , Histidina/química , Humanos , Hidrólise , Hipóxia/metabolismo , Camundongos , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Consumo de Oxigênio , Conformação Proteica , Subunidades ProteicasRESUMO
Correction for 'Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra' by Yamanappa Hunashal et al., Phys. Chem. Chem. Phys., 2020, 22, 6247-6259, DOI: .
RESUMO
The effect of extrinsic paramagnetic probes on NMR relaxation rates for surface mapping of proteins and other biopolymers is a widely investigated and powerful NMR technique. Here we describe a new application of those probes. It relies on the setting of the relaxation delay to generate magnetization equilibrium and off-equilibrium conditions, in order to tailor the extent of steady state signal recovery with and without the water-soluble nitroxide Tempol. With this approach it is possible to identify signals whose relaxation is affected by exchange processes and, from the relative assignments, to map the protein residues involved in association or conformational interconversion processes on a micro-to-millisecond time scale. This finding is confirmed by the comparison with the results obtained from relaxation dispersion measurements. This simple and convenient method allows preliminary inspection to highlight regions where structural or chemical exchange events are operative, in order to focus on quantitative subsequent determinations by transverse relaxation dispersion experiments or analogous NMR relaxation studies, and/or to gain insights into the predictions of calculations.
Assuntos
Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Magnetismo , Conformação ProteicaRESUMO
BACKGROUND: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, ß2-microglobulin (ß2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit ß2m fibril formation in vitro. METHODS: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on ß2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions. RESULTS: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of ß2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition. CONCLUSIONS: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions.
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Espectroscopia de Ressonância Magnética/métodos , Nanopartículas/química , Proteínas/química , Microglobulina beta-2/química , Amiloide/química , Óxidos N-Cíclicos/farmacologia , Dimerização , Espectroscopia de Ressonância de Spin Eletrônica , Ouro/química , Nanopartículas Metálicas/química , Modelos Moleculares , Domínios Proteicos , Mapeamento de Interação de Proteínas , Espectrofotometria , Marcadores de SpinRESUMO
Entropy calculation is an important step in the postprocessing of molecular dynamics trajectories or predictive models. In recent years the nearest neighbor method has emerged as a powerful method to deal in a flexible way with the dimensionality of the problem. Here we provide two programs, PBD2ENTROPY and PDB2TRENT that compute the conformational and translational-rotational entropy, respectively, based on the nearest neighbor method. PDB2ENTROPY takes in input two files containing the following: (1) conformational ensembles of the same molecule(s) in PDB format and (2) definitions of torsion angles (a default file is provided where additional user definitions can be easily implemented). PDB2TRENT takes in a file containing samples of the complexed molecules, a string specifying atoms providing the reference framework to superimpose samples, and a string specifying atoms used to compute rotation and translation of one molecule with respect to the other. The C programs and sample demonstration data are available on the GitHub repository (URL: http://github.com/federico-fogolari/pdb2entropy and http://github.com/federico-fogolari/pdb2trent ).
Assuntos
Simulação por Computador , Entropia , Modelos Moleculares , Benzeno/química , Conformação Molecular , Muramidase/química , Rotação , Software , Solventes/químicaRESUMO
We describe a new algorithmic approach able to automatically pick and track the NMR resonances of a large number of 2D NMR spectra acquired during a stepwise variation of a physical parameter. The method has been named Trace in Track (TINT), referring to the idea that a gaussian decomposition traces peaks within the tracks recognised through 3D mathematical morphology. It is capable of determining the evolution of the chemical shifts, intensity and linewidths of each tracked peak.The performances obtained in term of track reconstruction and correct assignment on realistic synthetic spectra were high above 90% when a noise level similar to that of experimental data were considered. TINT was applied successfully to several protein systems during a temperature ramp in isotope exchange experiments. A comparison with a state-of-the-art algorithm showed promising results for great numbers of spectra and low signal to noise ratios, when the graduality of the perturbation is appropriate. TINT can be applied to different kinds of high throughput chemical shift mapping experiments, with quasi-continuous variations, in which a quantitative automated recognition is crucial.
Assuntos
Espectroscopia de Ressonância Magnética , Modelos Teóricos , Proteínas/química , Algoritmos , Automação , Humanos , Espectroscopia de Ressonância Magnética/métodos , Reprodutibilidade dos TestesRESUMO
Mitochondria not only play a fundamental role in heart physiology but are also key effectors of dysfunction and death. This dual role assumes a new meaning after recent advances on the nature and regulation of the permeability transition pore, an inner membrane channel whose opening requires matrix Ca(2+) and is modulated by many effectors including reactive oxygen species, matrix cyclophilin D, Pi (inorganic phosphate), and matrix pH. The recent demonstration that the F-ATP synthase can reversibly undergo a Ca(2+)-dependent transition to form a channel that mediates the permeability transition opens new perspectives to the field. These findings demand a reassessment of the modifications of F-ATP synthase that take place in the heart under pathological conditions and of their potential role in determining the transition of F-ATP synthase from and energy-conserving into an energy-dissipating device.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Humanos , Mitocôndrias Cardíacas/fisiologia , Membranas Mitocondriais/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Miocárdio/metabolismo , PermeabilidadeRESUMO
The hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP allosterically modulates HCN through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN, to which cAMP binds. Although the apo versus holo conformational changes of the cAMP-binding domain (CBD) have been previously mapped, only limited information is currently available on the HCN IR dynamics, which have been hypothesized to play a critical role in the cAMP-dependent gating of HCN. Here, using molecular dynamics simulations validated and complemented by experimental NMR and CD data, we comparatively analyze HCN IR dynamics in the four states of the thermodynamic cycle arising from the coupling between cAMP binding and tetramerization equilibria. This extensive set of molecular dynamics trajectories captures the active-to-inactive transition that had remained elusive for other CBDs, and it provides unprecedented insight on the role of IR dynamics in HCN autoinhibition and its release by cAMP. Specifically, the IR tetramerization domain becomes more flexible in the monomeric states, removing steric clashes that the apo-CDB structure would otherwise impose. Furthermore, the simulations reveal that the active/inactive structural transition for the apo-monomeric CBD occurs through a manifold of pathways that are more divergent than previously anticipated. Upon cAMP binding, these pathways become disallowed, pre-confining the CBD conformational ensemble to a tetramer-compatible state. This conformational confinement primes the IR for tetramerization and thus provides a model of how cAMP controls HCN channel gating.
Assuntos
AMP Cíclico/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Multimerização Proteica , TermodinâmicaRESUMO
Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Bovinos , Linhagem Celular Tumoral , Dimerização , Humanos , Hidrólise , Potenciais da Membrana , Camundongos , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
The generalized Born model in the Onufriev, Bashford, and Case (Onufriev et al., Proteins: Struct Funct Genet 2004, 55, 383) implementation has emerged as one of the best compromises between accuracy and speed of computation. For simulations of nucleic acids, however, a number of issues should be addressed: (1) the generalized Born model is based on a linear model and the linearization of the reference Poisson-Boltmann equation may be questioned for highly charged systems as nucleic acids; (2) although much attention has been given to potentials, solvation forces could be much less sensitive to linearization than the potentials; and (3) the accuracy of the Onufriev-Bashford-Case (OBC) model for nucleic acids depends on fine tuning of parameters. Here, we show that the linearization of the Poisson Boltzmann equation has mild effects on computed forces, and that with optimal choice of the OBC model parameters, solvation forces, essential for molecular dynamics simulations, agree well with those computed using the reference Poisson-Boltzmann model.
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DNA/química , Proteínas/química , Modelos Químicos , Modelos Moleculares , TermodinâmicaRESUMO
The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein ß2-microglobulin (ß2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aß2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aß2m. From analysis of the NMR spectra collected at various R3Aß2m to αB-crystallin molar subunit ratios, it is concluded that the structured ß-sheet core and the apical loops of R3Aß2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type ß2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type ß2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated ß2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing ß2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.
Assuntos
Cadeia B de alfa-Cristalina/química , Microglobulina beta-2/química , Amiloide/química , Benzotiazóis , Corantes Fluorescentes/química , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estabilidade Proteica , Espectrometria de Massas por Ionização por Electrospray , Tiazóis/químicaRESUMO
Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human ß2-microglobulin (ß2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N ß2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type ß2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.
Assuntos
Amiloide/química , Mutação de Sentido Incorreto , Dobramento de Proteína , alfa-Cristalinas/química , Microglobulina beta-2/química , Substituição de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Amiloidose Familiar/genética , Amiloidose Familiar/metabolismo , Humanos , Estrutura Quaternária de Proteína , alfa-Cristalinas/genética , alfa-Cristalinas/metabolismo , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
OBJECTIVE: To investigate the polymorphisms in the promoter region of the B lymphocyte stimulator (BLyS) gene as markers of response to rituximab (RTX) in rheumatoid arthritis (RA). METHODS: The study was first conducted in 152 Italian RA patients and then replicated in an additional 117 RA patients (73 Italian, 44 British). The European League Against Rheumatism response criteria were used to evaluate the response rate at months 4 and 6 after the first cycle of RTX, by means of the Disease Activity Score in 28 joints using the erythrocyte sedimentation rate; patients were classified according to the best response shown between months 4 and 6. BLyS promoter polymorphisms were analyzed by polymerase chain reaction followed by the analysis of the restriction fragments, BLyS promoter haplotypes were analyzed using the expectation-maximization algorithm, and BLyS serum levels were analyzed using enzyme-linked immunosorbent assay. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs). RESULTS: The TTTT BLyS promoter haplotype appeared to be significantly associated with response to RTX only in the subset of seropositive patients (those positive for rheumatoid factor and/or anti-cyclic citrullinated peptide). The replication study confirmed that this association was limited to seropositive RA patients in whom treatment with anti-tumor necrosis factor (anti-TNF) agents had previously failed. In the whole series of seropositive patients in whom anti-TNF agents had previously failed, patients carrying the TTTT BLyS promoter haplotype were more prevalent in good responders (18 of 43 [41.9%]) than in moderate responders (20 of 83 [24.1%]) or in nonresponders (1 of 21 [4.8%]) (for good responders versus nonresponders, OR 14.4 [95% CI 1.77-117.39], P=0.0028). Furthermore, multivariate analysis selected the TTTT BLyS promoter haplotype as an independent marker of good response to RTX (for good responders versus nonresponders, OR 16.2 [95% CI 1.7-152.5], P=0.01; for good responders versus moderate responders and nonresponders combined, OR 3.1 [95% CI 1.2-7.8], P=0.02). The relationship between BLyS polymorphisms and BLyS serum levels remained unclear. CONCLUSION: BLyS promoter genotyping may be suitable for identifying seropositive RA patients who may have a good response to RTX after anti-TNF agents have failed.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Fator Ativador de Células B/genética , Fator de Necrose Tumoral alfa/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/genética , Sedimentação Sanguínea , Estudos de Coortes , Resistência a Medicamentos/genética , Inglaterra , Ensaio de Imunoadsorção Enzimática , Feminino , Haplótipos , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Estudos Retrospectivos , Rituximab , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto JovemRESUMO
The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial F(O)F1 ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F1 sector, it makes stable contacts with both F1 and the peripheral stalk, ensuring the structural and functional coupling between F(O) and F1, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca²âº required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca²âº-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Adenosina Trifosfatases/análise , Animais , Proteínas de Transporte/análise , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Humanos , Proteínas de Membrana/análise , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/análise , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.