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1.
Sci Rep ; 10(1): 14290, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868801

RESUMO

Several drug candidates have been proposed and tested as the latest clinical treatment for coronavirus pneumonia (COVID-19). Chloroquine, hydroxychloroquine, ritonavir/lopinavir, and favipiravir are under trials for the treatment of this disease. The hyperpolarization technique has the ability to further provide a better understanding of the roles of these drugs at the molecular scale and in different applications in the field of nuclear magnetic resonance/magnetic resonance imaging. This technique may provide new opportunities in diagnosis and research of COVID-19. Signal amplification by reversible exchange-based hyperpolarization studies on large-sized drug candidates were carried out. We observed hyperpolarized proton signals from whole structures, due to the unprecedented long-distance polarization transfer by para-hydrogen. We also found that the optimal magnetic field for the maximum polarization transfer yield was dependent on the molecular structure. We can expect further research on the hyperpolarization of other important large molecules, isotope labeling, as well as polarization transfer on nuclei with a long spin relaxation time. A clinical perspective of these features on drug molecules can broaden the application of hyperpolarization techniques for therapeutic studies.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/virologia , Descoberta de Drogas , Pneumonia Viral/virologia , Amidas/química , Amidas/farmacologia , Antivirais/química , Cloroquina/química , Cloroquina/farmacologia , Infecções por Coronavirus/diagnóstico , Descoberta de Drogas/métodos , Humanos , Lopinavir/química , Lopinavir/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Pandemias , Pneumonia Viral/diagnóstico , Pirazinas/química , Pirazinas/farmacologia , Ritonavir/química , Ritonavir/farmacologia
2.
PLoS Biol ; 18(9): e3000848, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898131

RESUMO

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Miocárdio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Sarcômeros/metabolismo
3.
PLoS One ; 15(7): e0235925, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639967

RESUMO

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.


Assuntos
Síndrome de Angelman/genética , Ubiquitina-Proteína Ligases/metabolismo , Síndrome de Angelman/patologia , Biocatálise , Dicroísmo Circular , Humanos , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
4.
Nat Commun ; 11(1): 3656, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694517

RESUMO

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.


Assuntos
Proteínas Aviárias/ultraestrutura , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas Nucleares/ultraestrutura , Domínios Proteicos/genética , RNA Replicase/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Virais/ultraestrutura , Animais , Proteínas Aviárias/metabolismo , Aves/virologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , RNA Replicase/genética , RNA Replicase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
5.
J Asian Nat Prod Res ; 22(10): 895-904, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32536209

RESUMO

Two new grayanane-type (1 and 2) and one new kalmane-type diterpenoids (3), together with 16 known compounds, were isolated from the roots of Rhododendron micranthum. The structures of new compounds were fully determined on the basis of spectroscopic analysis, including HRESIMS, 1 D and 2 D NMR data. An acetic acid-induced writhing test in mice was proceeded to evaluate the antinociceptive activities of compounds 1-3, 5-6, 9-14 and 16. Compared to vehicle-injected mice, compounds 1, 6, 14 and 16 showed significant antinociceptive effects with writhe inhibition rates of 45.8%-64.2% at a dose of 0.1 mg/kg, and compounds 10, 12 and 13 showed significant antinociceptive effects with writhe inhibition rates of 33.9%-64.8% at a dose of 5 mg/kg. Compound 2 showed potent antinociceptive effects with writhe inhibition rates of 86.1% and 54.7% at doses of 8 mg/kg and 0.8 mg/kg, respectively. [Formula: see text].


Assuntos
Diterpenos , Rhododendron , Analgésicos , Animais , Cristalografia por Raios X , Camundongos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular
6.
Nat Commun ; 11(1): 3146, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561718

RESUMO

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Biossíntese de Proteínas/fisiologia , Capuzes de RNA/metabolismo , Biologia Computacional , Fator de Iniciação 4E em Eucariotos/genética , Proteínas Intrinsicamente Desordenadas/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional/fisiologia
7.
Nat Commun ; 11(1): 3137, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561731

RESUMO

The close synergy between peptides and nucleic acids in current biology is suggestive of a functional co-evolution between the two polymers. Here we show that cationic proto-peptides (depsipeptides and polyesters), either produced as mixtures from plausibly prebiotic dry-down reactions or synthetically prepared in pure form, can engage in direct interactions with RNA resulting in mutual stabilization. Cationic proto-peptides significantly increase the thermal stability of folded RNA structures. In turn, RNA increases the lifetime of a depsipeptide by >30-fold. Proto-peptides containing the proteinaceous amino acids Lys, Arg, or His adjacent to backbone ester bonds generally promote RNA duplex thermal stability to a greater magnitude than do analogous sequences containing non-proteinaceous residues. Our findings support a model in which tightly-intertwined biological dependencies of RNA and protein reflect a long co-evolutionary history that began with rudimentary, mutually-stabilizing interactions at early stages of polypeptide and nucleic acid co-existence.


Assuntos
Evolução Molecular , Peptídeos/metabolismo , Dobramento de Proteína , Estabilidade de RNA , RNA/metabolismo , Sequência de Aminoácidos , Aminobutiratos/química , Aminobutiratos/metabolismo , Cátions/química , Cátions/metabolismo , Dicroísmo Circular , Hidrólise , Ressonância Magnética Nuclear Biomolecular , Origem da Vida , Ornitina/química , Ornitina/metabolismo , Peptídeos/química , Estabilidade Proteica , RNA/química , beta-Alanina/análogos & derivados , beta-Alanina/química , beta-Alanina/metabolismo
8.
Nat Commun ; 11(1): 2830, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503980

RESUMO

The Spitzenkörper (SPK) constitutes a collection of secretory vesicles and polarity-related proteins intimately associated with polarized growth of fungal hyphae. Many SPK-localized proteins are known, but their assembly and dynamics remain poorly understood. Here, we identify protein-protein interaction cascades leading to assembly of two SPK scaffolds and recruitment of diverse effectors in Neurospora crassa. Both scaffolds are transported to the SPK by the myosin V motor (MYO-5), with the coiled-coil protein SPZ-1 acting as cargo adaptor. Neither scaffold appears to be required for accumulation of SPK secretory vesicles. One scaffold consists of Leashin-2 (LAH-2), which is required for SPK localization of the signalling kinase COT-1 and the glycolysis enzyme GPI-1. The other scaffold comprises a complex of Janus-1 (JNS-1) and the polarisome protein SPA-2. Via its Spa homology domain (SHD), SPA-2 recruits a calponin domain-containing F-actin effector (CCP-1). The SHD NMR structure reveals a conserved surface groove required for effector binding. Similarities between SPA-2/JNS-1 and the metazoan GIT/PIX complex identify foundational features of the cell polarity apparatus that predate the fungal-metazoan divergence.


Assuntos
Polaridade Celular , Proteínas Fúngicas/metabolismo , Miosina Tipo V/metabolismo , Neurospora crassa/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Fúngicas/química , Hifas/citologia , Hifas/metabolismo , Miosina Tipo V/química , Neurospora crassa/citologia , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Mapas de Interação de Proteínas
9.
PLoS One ; 15(5): e0232338, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32421702

RESUMO

End-binding proteins (EBs) associate with the growing microtubule plus ends to regulate microtubule dynamics as well as the interaction with intracellular structures. EB3 contributes to pathological vascular leakage through interacting with the inositol 1,4,5-trisphosphate receptor 3 (IP3R3), a calcium channel located at the endoplasmic reticulum membrane. The C-terminal domain of EB3 (residues 200-281) is functionally important for this interaction because it contains the effector binding sites, a prerequisite for EB3 activity and specificity. Structural data for this domain is limited. Here, we report the backbone chemical shift assignments for the human EB3 C-terminal domain and computationally explore its EB3 conformations. Backbone assignments, along with computational models, will allow future investigation of EB3 structural dynamics, interactions with effectors, and will facilitate the development of novel EB3 inhibitors.


Assuntos
Proteínas Associadas aos Microtúbulos/química , Ressonância Magnética Nuclear Biomolecular , Humanos , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína
10.
PLoS Comput Biol ; 16(5): e1007877, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32401799

RESUMO

Experimental chemical shifts (CS) from solution and solid state magic-angle-spinning nuclear magnetic resonance (NMR) spectra provide atomic level information for each amino acid within a protein or protein complex. However, structure determination of large complexes and assemblies based on NMR data alone remains challenging due to the complexity of the calculations. Here, we present a hardware accelerated strategy for the estimation of NMR chemical-shifts of large macromolecular complexes based on the previously published PPM_One software. The original code was not viable for computing large complexes, with our largest dataset taking approximately 14 hours to complete. Our results show that serial code refactoring and parallel acceleration brought down the time taken of the software running on an NVIDIA Volta 100 (V100) Graphic Processing Unit (GPU) to 46.71 seconds for our largest dataset of 11.3 million atoms. We use OpenACC, a directive-based programming model for porting the application to a heterogeneous system consisting of x86 processors and NVIDIA GPUs. Finally, we demonstrate the feasibility of our approach in systems of increasing complexity ranging from 100K to 11.3M atoms.


Assuntos
Biologia Computacional , Conformação Proteica , Conjuntos de Dados como Assunto , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Reprodutibilidade dos Testes
11.
Nucleic Acids Res ; 48(W1): W41-W47, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32383755

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy is a method of choice to study the dynamics and determine the atomic structure of macromolecules in solution. The standalone program ARIA (Ambiguous Restraints for Iterative Assignment) for automated assignment of nuclear Overhauser enhancement (NOE) data and structure calculation is well established in the NMR community. To ultimately provide a perfectly transparent and easy to use service, we designed an online user interface to ARIA with additional functionalities. Data conversion, structure calculation setup and execution, followed by interactive visualization of the generated 3D structures are all integrated in ARIAweb and freely accessible at https://ariaweb.pasteur.fr.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Software , Animais , Humanos , Camundongos , Modelos Moleculares , RNA/química
12.
Nucleic Acids Res ; 48(W1): W36-W40, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32459331

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy data provides valuable information on the behaviour of proteins in solution. The primary data to determine when studying proteins are the per-atom NMR chemical shifts, which reflect the local environment of atoms and provide insights into amino acid residue dynamics and conformation. Within an amino acid residue, chemical shifts present multi-dimensional and complexly cross-correlated information, making them difficult to analyse. The ShiftCrypt method, based on neural network auto-encoder architecture, compresses the per-amino acid chemical shift information in a single, interpretable, amino acid-type independent value that reflects the biophysical state of a residue. We here present the ShiftCrypt web server, which makes the method readily available. The server accepts chemical shifts input files in the NMR Exchange Format (NEF) or NMR-STAR format, executes ShiftCrypt and visualises the results, which are also accessible via an API. It also enables the "biophysically-based" pairwise alignment of two proteins based on their ShiftCrypt values. This approach uses Dynamic Time Warping and can optionally include their amino acid code information, and has applications in, for example, the alignment of disordered regions. The server uses a token-based system to ensure the anonymity of the users and results. The web server is available at www.bio2byte.be/shiftcrypt.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Software , Aminoácidos/química , Redes Neurais de Computação , Desnaturação Proteica , Dobramento de Proteína , Desdobramento de Proteína
13.
Proc Natl Acad Sci U S A ; 117(23): 12836-12846, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457157

RESUMO

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)-based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics. We illustrate the methodology with studies of the 153-base pair Widom DNA molecule that is simultaneously methyl-labeled at five sites, showing that high-quality 13C-1H spectra can be recorded on 100 µM samples in a few minutes. NMR spin relaxation studies of labeled methyl groups in both DNA and the H2B histone protein component of the 200-kDa nucleosome core particle (NCP) establish that methyl groups at 5mC and 6mA positions are, in general, more rigid than Ile, Leu, and Val methyl probes in protein side chains. Studies focusing on histone H2B of NCPs wrapped with either wild-type DNA or DNA methylated at all 26 CpG sites highlight the utility of NMR in investigating the structural dynamics of the NCP and how its histone core is affected through DNA methylation, an important regulator of transcription.


Assuntos
DNA/ultraestrutura , Ressonância Magnética Nuclear Biomolecular/métodos , Nucleossomos/ultraestrutura , Análise Espectral/métodos , Adenina/química , Isótopos de Carbono , Ilhas de CpG , Citosina/química , DNA/química , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Simulação de Dinâmica Molecular , Peso Molecular
14.
Chemistry ; 26(30): 6789-6792, 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32240561

RESUMO

Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time 31 P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.


Assuntos
Diacilglicerol Quinase/metabolismo , Escherichia coli/metabolismo , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/metabolismo , Catálise , Fenômenos Fisiológicos Celulares , Diacilglicerol Quinase/química , Escherichia coli/química , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosforilação
15.
Science ; 368(6489): 413-417, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32327595

RESUMO

Heterogeneous transcriptional start site usage by HIV-1 produces 5'-capped RNAs beginning with one, two, or three 5'-guanosines (Cap1G, Cap2G, or Cap3G, respectively) that are either selected for packaging as genomes (Cap1G) or retained in cells as translatable messenger RNAs (mRNAs) (Cap2G and Cap3G). To understand how 5'-guanosine number influences fate, we probed the structures of capped HIV-1 leader RNAs by deuterium-edited nuclear magnetic resonance. The Cap1G transcript adopts a dimeric multihairpin structure that sequesters the cap, inhibits interactions with eukaryotic translation initiation factor 4E, and resists decapping. The Cap2G and Cap3G transcripts adopt an alternate structure with an elongated central helix, exposed splice donor residues, and an accessible cap. Extensive remodeling, achieved at the energetic cost of a G-C base pair, explains how a single 5'-guanosine modifies the function of a ~9-kilobase HIV-1 transcript.


Assuntos
Pareamento de Bases , Regulação Viral da Expressão Gênica , HIV-1/genética , Capuzes de RNA/genética , RNA Viral/genética , Sítio de Iniciação de Transcrição , Regiões 5' não Traduzidas/genética , Composição de Bases , Fator de Iniciação 4E em Eucariotos/metabolismo , Guanosina/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Biossíntese de Proteínas , Capuzes de RNA/química , RNA Mensageiro/genética
16.
PLoS One ; 15(4): e0232015, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330166

RESUMO

Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.


Assuntos
Conformação Proteica , Proteínas/metabolismo , Ribossomos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica/fisiologia , Estabilidade Proteica , Ribonuclease Pancreático/metabolismo
17.
J Phys Chem Lett ; 11(9): 3384-3389, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32286073

RESUMO

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where 1HN spectra cannot be obtained. The approach uses a weak 1H B1 field, searching for elusive 1HN resonance frequencies that become encoded in the intensities of cross-peaks in three-dimensional 1Hα-detect spectra. Applications to the CAPRIN1 protein in both dilute- and phase-separated states highlight the utility of the method, establishing that accurate 1HN chemical shifts can be obtained even in cases where solvent hydrogen exchange rates are on the order of 1500 s-1.


Assuntos
Proteínas de Ciclo Celular/química , Hidrogênio/química , Proteínas Intrinsicamente Desordenadas/química , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular
18.
J Med Chem ; 63(10): 5367-5386, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32342688

RESUMO

In search of novel drugs against tuberculosis, we previously discovered and profiled a novel hydantoin-based family that demonstrated highly promising in vitro potency against Mycobacterium. tuberculosis. The compounds were found to be noncovalent inhibitors of DprE1, a subunit of decaprenylphosphoryl-ß-d-ribose-2'-epimerase. This protein, localized in the periplasmic space of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug target. Here, we report the further SAR exploration of this chemical family through more than 80 new analogues. Among these, the most active representatives combined submicromolar cellular potency and nanomolar target affinity with balanced physicochemical properties and low human cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family of DprE1 inhibitors represents a promising noncovalent lead series for the discovery of novel antituberculosis agents.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Hidantoínas/química , Hidantoínas/farmacologia , Oxirredutases do Álcool/metabolismo , Animais , Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Feminino , Células Hep G2 , Humanos , Hidantoínas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo
19.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 326-331, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254056

RESUMO

The study of ion channels dates back to the 1950s and the groundbreaking electrophysiology work of Hodgin and Huxley, who used giant squid axons to probe how action potentials in neurons were initiated and propagated. More recently, several experiments using different structural biology techniques and approaches have been conducted to try to understand how potassium ions permeate through the selectivity filter of potassium ion channels. Two mechanisms of permeation have been proposed, and each of the two mechanisms is supported by different experiments. The key structural biology experiments conducted so far to try to understand how ion permeation takes place in potassium ion channels are reviewed and discussed. Protein crystallography has made, and continues to make, key contributions in this field, often through the use of anomalous scattering. Other structural biology techniques used to study the contents of the selectivity filter include solid-state nuclear magnetic resonance and two-dimensional infrared spectroscopy, both of which make clever use of isotopic labeling techniques, while molecular-dynamics simulations of ion flow through the selectivity filter have been enabled by the growing number of potassium ion channel structures deposited in the Protein Data Bank.


Assuntos
Simulação de Dinâmica Molecular , Canais de Potássio/química , Potássio/química , Cristalografia por Raios X , Marcação por Isótopo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Espalhamento de Radiação , Espectrofotometria Infravermelho
20.
Clín. investig. arterioscler. (Ed. impr.) ; 32(2): 49-58, mar.-abr. 2020. tab, graf
Artigo em Inglês | IBECS | ID: ibc-187146

RESUMO

Background and aims: The first line of therapy in children with hypercholesterolaemia is therapeutic lifestyle changes (TLSC). The efficacy of lifestyle intervention in children with familial hypercholesterolaemia (FH), where LDL-C levels are genetically driven, deserves a focused study. Aims: To evaluate the impact of a lifestyle education program, focused on food patterns and physical activity, on lipid profiles assessed by nuclear magnetic resonance (NMR) in children with FH vs. non-FH. Methods: Phase 1 was a cross-sectional study of baseline characteristics, and phase 2 was a prospective TLSC intervention study. In total, the study included 238 children (4 to 18 years old; 47% girls) attending the lipid unit of our hospital due to high cholesterol levels. Eighty-five were diagnosed with FH (72% genetic positive), and 153 were diagnosed with non-Familial hypercholesterolaemia. A quantitative food frequency questionnaire (FFQ) including 137 items was used. Physical activity (PA) was assessed by the Minnesota questionnaire. The lipid profile was assessed using the 2D-1H-NMR (Liposcale test). A total of 127 children (81 in the FH group) participated in the prospective phase and were re-assessed after 1 year of the TLSC intervention, consisting of education on lifestyle changes delivered by a specialized nutritionist. Results: The FH and non-FH groups were similar in anthropometry and clinical data, except that those in the FH were slightly younger than those in the non-FH group. Both the FH and non-FH groups showed a similar diet composition characterized by a high absolute calorie intake and a high percentage of fat, mainly saturated fat. The PA was below the recommended level in both groups. After one year of TLSC, the percentage of total and saturated fats was reduced, and the amount of fiber increased significantly in both groups. The percentage of protein increased slightly. The number of children engaged in at least 1 hour/day of PA increased by 56% in the FH group and by 53% in the non-FH group, and both these increases were significant. The total and small-LDL particle numbers were reduced in both groups, although the absolute change was greater in the FH group than in the non-FH group. Conclusions: Educational strategies to implement TLSC in children lead to empowerment, increased adherence, and overall metabolic improvement in children with high blood cholesterol, including those with FH


Antecedentes y objetivos: La primera línea de terapia en niños con hipercolesterolemia son los cambios terapéuticos en el estilo de vida (TLSC). La eficacia de la intervención en el estilo de vida en niños con hipercolesterolemia familiar (HF), en los que los niveles de LDL-C son generados genéticamente, merece un estudio específico. Objetivos: Evaluar el impacto de un programa de educación sobre el estilo de vida, centrado en los patrones alimentarios y la actividad física, sobre el perfil lipídico evaluado por resonancia magnética nuclear (RMN) en niños con HF versus no HF. Métodos: La fase 1 fue un estudio transversal de las características basales, y la fase 2 fue un estudio prospectivo de intervención mediante TLSC. En total, el estudio incluyó a 238 niños (de 4 a 18 años; 47% niñas) que asistieron a la unidad de lípidos de nuestro hospital debido a los altos niveles de colesterol. Ochenta y cinco fueron diagnosticados con HF (72% genéticamente positivos), y 153 fueron diagnosticados de no HF. Se utilizó un cuestionario cuantitativo de frecuencia de alimentos (FFQ) que incluye 137 ítems. La actividad física (AF) se evaluó mediante el cuestionario de Minnesota. El perfil lipídico se evaluó mediante la prueba 2D-1H-NMR (Liposcale Test). Un total de 127 niños (81 en el grupo HF) participaron en la fase prospectiva y fueron reevaluados después de 1 año de la intervención mediante TLSC, que consistió en educación sobre cambios en el estilo de vida impartida por una nutricionista especializada. Resultados: Los grupos HF y no HF fueron similares en los datos antropométricos y clínicos, excepto que los HF eran ligeramente más jóvenes que los no HF. Los participantes de ambos grupos mostraron una composición de dieta similar caracterizada por un alto consumo de calorías totales y un alto porcentaje de grasas, principalmente grasas saturadas. La AF estuvo por debajo del nivel recomendado en ambos grupos. Después de un año de TLSC, se redujo el porcentaje de grasas totales y saturadas, y la cantidad de fibra aumentó significativamente en ambos grupos. El porcentaje de proteína aumentó ligeramente. El número de niños involucrados en al menos 1 hora/día de AF aumentó en un 56% en el grupo de HF y en un 53% en el grupo sin HF, y ambos aumentos fueron significativos. Los números de partículas LDL totales y pequeñas se redujeron en ambos grupos, aunque el cambio absoluto fue mayor en el grupo HF que en el grupo no HF. Conclusiones: Las estrategias educativas para implementar TLSC en niños conducen al empoderamiento, al aumento de la adherencia y a la mejora metabólica general en niños con colesterol alto en sangre, incluidos aquellos con HF


Assuntos
Humanos , Masculino , Feminino , Pré-Escolar , Criança , Adolescente , Hipercolesterolemia/sangue , Hipercolesterolemia/terapia , Estilo de Vida , Educação de Pacientes como Assunto , Lipoproteínas LDL/sangue , Dietoterapia , Terapia por Exercício , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/terapia , Ressonância Magnética Nuclear Biomolecular , Estudos Transversais , Estudos Prospectivos , Resultado do Tratamento
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