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NMR is widely considered the gold standard for organic compound structure determination. As such, NMR is routinely used in organic compound identification, drug metabolite characterization, natural product discovery, and the deconvolution of metabolite mixtures in biofluids (metabolomics and exposomics). In many cases, compound identification by NMR is achieved by matching measured NMR spectra to experimentally collected NMR spectral reference libraries. Unfortunately, the number of available experimental NMR reference spectra, especially for metabolomics, medical diagnostics, or drug-related studies, is quite small. This experimental gap could be filled by predicting NMR chemical shifts for known compounds using computational methods such as machine learning (ML). Here, we describe how a deep learning algorithm that is trained on a high-quality, "solvent-aware" experimental dataset can be used to predict 1H chemical shifts more accurately than any other known method. The new program, called PROSPRE (PROton Shift PREdictor) can accurately (mean absolute error of <0.10 ppm) predict 1H chemical shifts in water (at neutral pH), chloroform, dimethyl sulfoxide, and methanol from a user-submitted chemical structure. PROSPRE (pronounced "prosper") has also been used to predict 1H chemical shifts for >600,000 molecules in many popular metabolomic, drug, and natural product databases.
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We show that the nucleic acid bases adenine, cytosine, guanine, thymine, and uracil, as well as 2,6-diaminopurine, and the "core" nucleic acid bases purine and pyrimidine, are stable for more than one year in concentrated sulfuric acid at room temperature and at acid concentrations relevant for Venus clouds (81% w/w to 98% w/w acid, the rest water). This work builds on our initial stability studies and is the first ever to test the reactivity and structural integrity of organic molecules subjected to extended incubation in concentrated sulfuric acid. The one-year-long stability of nucleic acid bases supports the notion that the Venus cloud environment-composed of concentrated sulfuric acid-may be able to support complex organic chemicals for extended periods of time.
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Nuclear magnetic resonance (NMR) data are rarely deposited in open databases, leading to loss of critical scientific knowledge. Existing data reporting methods (images, tables, lists of values) contain less information than raw data and are poorly standardized. Together, these issues limit FAIR (findable, accessible, interoperable, reusable) access to these data, which in turn creates barriers for compound dereplication and the development of new data-driven discovery tools. Existing NMR databases either are not designed for natural products data or employ complex deposition interfaces that disincentivize deposition. Journals, including the Journal of Natural Products (JNP), are now requiring data submission as part of the publication process, creating the need for a streamlined, user-friendly mechanism to deposit and distribute NMR data.
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Productos Biológicos , Bases de Datos Factuales , Espectroscopía de Resonancia MagnéticaRESUMEN
Nuclear magnetic resonance (NMR) spectral analysis of biofluids can be a time-consuming process, requiring the expertise of a trained operator. With NMR becoming increasingly popular in the field of metabolomics, there is a growing need to change this paradigm and to automate the process. Here we introduce MagMet, an online web server, that automates the processing and quantification of 1D 1 H NMR spectra from biofluids-specifically, human serum/plasma metabolites, including those associated with inborn errors of metabolism (IEM). MagMet uses a highly efficient data processing procedure that performs automatic Fourier Transformation, phase correction, baseline optimization, chemical shift referencing, water signal removal, and peak picking/peak alignment. MagMet then uses the peak positions, linewidth information, and J-couplings from its own specially prepared standard metabolite reference spectral NMR library of 85 serum/plasma compounds to identify and quantify compounds from experimentally acquired NMR spectra of serum/plasma. MagMet employs linewidth adjustment for more consistent quantification of metabolites from higher field instruments and incorporates a highly efficient data processing procedure for more rapid and accurate detection and quantification of metabolites. This optimized algorithm allows the MagMet webserver to quickly detect and quantify 58 serum/plasma metabolites in 2.6 min per spectrum (when processing a dataset of 50-100 spectra). MagMet's performance was also assessed using spectra collected from defined mixtures (simulating other biofluids), with >100 previously measured plasma spectra, and from spiked serum/plasma samples simulating known IEMs. In all cases, MagMet performed with precision and accuracy matching the performance of human spectral profiling experts. MagMet is available at http://magmet.ca.
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Imagen por Resonancia Magnética , Metabolómica , Humanos , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Suero , AlgoritmosRESUMEN
We report the development of a software program, called MagMet-F, that automates the processing and quantification of 1D 1 H NMR of human fecal extracts. To optimize the program, we identified 82 potential fecal metabolites using 1D 1 H NMR of six human fecal extracts using manual profiling and a literature review of known fecal metabolites. We acquired pure versions of those metabolites and then acquired their 1D 1 H NMR spectra at 700 MHz to generate a fecal metabolite spectral library for MagMet-F. The fitting of these metabolites by MagMet-F was iteratively optimized to replicate manual profiling. We validated MagMet-F's automated profiling using a test set of six fecal extracts. It correctly identified 80% of the compounds and quantified those within <20% of the values determined by manual profiling using Chenomx. We also compared MagMet-F's profiling performance to two other open-access NMR profiling tools, Bayesil and Batman. MagMet-F outperformed both. Bayesil repeatedly overestimated metabolite concentrations by 10% to 40% while Batman was unable to properly quantify any compounds and took 10-20× longer. We have implemented MagMet-F as a freely accessible web server to enable automated, fast and convenient 1D 1 H NMR spectral profiling of fecal samples. MagMet-F is available at https://www.magmet.ca.
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Metabolómica , Programas Informáticos , Humanos , Metabolómica/métodos , Espectroscopía de Resonancia Magnética/métodos , Imagen por Resonancia MagnéticaRESUMEN
What constitutes a habitable planet is a frontier to be explored and requires pushing the boundaries of our terracentric viewpoint for what we deem to be a habitable environment. Despite Venus' 700 K surface temperature being too hot for any plausible solvent and most organic covalent chemistry, Venus' cloud-filled atmosphere layers at 48 to 60 km above the surface hold the main requirements for life: suitable temperatures for covalent bonds; an energy source (sunlight); and a liquid solvent. Yet, the Venus clouds are widely thought to be incapable of supporting life because the droplets are composed of concentrated liquid sulfuric acid-an aggressive solvent that is assumed to rapidly destroy most biochemicals of life on Earth. Recent work, however, demonstrates that a rich organic chemistry can evolve from simple precursor molecules seeded into concentrated sulfuric acid, a result that is corroborated by domain knowledge in industry that such chemistry leads to complex molecules, including aromatics. We aim to expand the set of molecules known to be stable in concentrated sulfuric acid. Here, we show that nucleic acid bases adenine, cytosine, guanine, thymine, and uracil, as well as 2,6-diaminopurine and the "core" nucleic acid bases purine and pyrimidine, are stable in sulfuric acid in the Venus cloud temperature and sulfuric acid concentration range, using UV spectroscopy and combinations of 1D and 2D 1H 13C 15N NMR spectroscopy. The stability of nucleic acid bases in concentrated sulfuric acid advances the idea that chemistry to support life may exist in the Venus cloud particle environment.
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Bivalvos , Venus , Adenina , Agresión , Ácidos SulfúricosRESUMEN
While NMR-based metabolomics is only about 20 years old, NMR has been a key part of metabolic and metabolism studies for >40 years. Historically, metabolic researchers used NMR because of its high level of reproducibility, superb instrument stability, facile sample preparation protocols, inherently quantitative character, non-destructive nature, and amenability to automation. In this chapter, we provide a short history of NMR-based metabolomics. We then provide a detailed description of some of the practical aspects of performing NMR-based metabolomics studies including sample preparation, pulse sequence selection, and spectral acquisition and processing. The two different approaches to metabolomics data analysis, targeted vs. untargeted, are briefly outlined. We also describe several software packages to help users process NMR spectra obtained via these two different approaches. We then give several examples of useful or interesting applications of NMR-based metabolomics, ranging from applications to drug toxicology, to identifying inborn errors of metabolism to analyzing the contents of biofluids from dairy cattle. Throughout this chapter, we will highlight the strengths and limitations of NMR-based metabolomics. Additionally, we will conclude with descriptions of recent advances in NMR hardware, methodology, and software and speculate about where NMR-based metabolomics is going in the next 5-10 years.
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Imagen por Resonancia Magnética , Metabolómica , Animales , Bovinos , Reproducibilidad de los Resultados , Metabolómica/métodos , Espectroscopía de Resonancia Magnética/métodosRESUMEN
The Human Metabolome Database or HMDB (https://hmdb.ca) has been providing comprehensive reference information about human metabolites and their associated biological, physiological and chemical properties since 2007. Over the past 15 years, the HMDB has grown and evolved significantly to meet the needs of the metabolomics community and respond to continuing changes in internet and computing technology. This year's update, HMDB 5.0, brings a number of important improvements and upgrades to the database. These should make the HMDB more useful and more appealing to a larger cross-section of users. In particular, these improvements include: (i) a significant increase in the number of metabolite entries (from 114 100 to 217 920 compounds); (ii) enhancements to the quality and depth of metabolite descriptions; (iii) the addition of new structure, spectral and pathway visualization tools; (iv) the inclusion of many new and much more accurately predicted spectral data sets, including predicted NMR spectra, more accurately predicted MS spectra, predicted retention indices and predicted collision cross section data and (v) enhancements to the HMDB's search functions to facilitate better compound identification. Many other minor improvements and updates to the content, the interface, and general performance of the HMDB website have also been made. Overall, we believe these upgrades and updates should greatly enhance the HMDB's ease of use and its potential applications not only in human metabolomics but also in exposomics, lipidomics, nutritional science, biochemistry and clinical chemistry.
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Bases de Datos Genéticas , Metaboloma/genética , Metabolómica/clasificación , Humanos , Lipidómica/clasificación , Espectrometría de Masas , Interfaz Usuario-ComputadorRESUMEN
The Natural Products Magnetic Resonance Database (NP-MRD) is a comprehensive, freely available electronic resource for the deposition, distribution, searching and retrieval of nuclear magnetic resonance (NMR) data on natural products, metabolites and other biologically derived chemicals. NMR spectroscopy has long been viewed as the 'gold standard' for the structure determination of novel natural products and novel metabolites. NMR is also widely used in natural product dereplication and the characterization of biofluid mixtures (metabolomics). All of these NMR applications require large collections of high quality, well-annotated, referential NMR spectra of pure compounds. Unfortunately, referential NMR spectral collections for natural products are quite limited. It is because of the critical need for dedicated, open access natural product NMR resources that the NP-MRD was funded by the National Institute of Health (NIH). Since its launch in 2020, the NP-MRD has grown quickly to become the world's largest repository for NMR data on natural products and other biological substances. It currently contains both structural and NMR data for nearly 41,000 natural product compounds from >7400 different living species. All structural, spectroscopic and descriptive data in the NP-MRD is interactively viewable, searchable and fully downloadable in multiple formats. Extensive hyperlinks to other databases of relevance are also provided. The NP-MRD also supports community deposition of NMR assignments and NMR spectra (1D and 2D) of natural products and related meta-data. The deposition system performs extensive data enrichment, automated data format conversion and spectral/assignment evaluation. Details of these database features, how they are implemented and plans for future upgrades are also provided. The NP-MRD is available at https://np-mrd.org.
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Productos Biológicos/química , Bases de Datos Factuales , Espectroscopía de Resonancia Magnética , Programas Informáticos , Productos Biológicos/clasificación , InternetRESUMEN
PRCIS: Modeling of visual field and pharmacy data (Kaiser Permanente, 2001 to 2014) from open-angle/pseudoexfoliation glaucoma patients in clinical practice indicated a significant inverse association between the level of medication adherence and rate of visual field progression. PURPOSE: The aim was to quantify the effect of nonadherence to topical hypotensive medication on glaucomatous visual field progression in clinical practice. METHODS: Retrospective analysis of combined visual field and pharmacy data from Kaiser Permanente Southern California's HealthConnect electronic health record database. Patients with a diagnosis of primary open-angle glaucoma or pseudoexfoliation glaucoma (2001 to 2011) and ≥3 subsequent visual field tests of the same Swedish Interactive Threshold Algorithm type were followed up from first medication fill to final visual field test. Medication adherence (proportion of days covered) was estimated from pharmacy refill data. A conditional growth model was used to estimate the effect of adherence level in modifying the progression of mean deviation over time after adjusting for potential confounders, including age, sex, race/ethnicity, baseline glaucoma severity, and comorbidity. RESULTS: In total, 6343 eligible patients were included in the study and followed for (mean) 5.8 years; average treatment adherence during follow-up was 73%. After controlling for confounders and the interaction between time and baseline disease severity, the model indicated that mean deviation progression was significantly (P=0.006) reduced by 0.006 dB per year for each 10% (absolute) increase in adherence. Model estimates of time to glaucoma progression (mean deviation change -3 dB from baseline) were 8.3 and 9.3 years for patients with adherence levels of 20% and 80%, respectively. CONCLUSIONS: Improving patient adherence to topical glaucoma medication may result in slower deterioration in visual function over time.
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Glaucoma de Ángulo Abierto , Progresión de la Enfermedad , Estudios de Seguimiento , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Humanos , Presión Intraocular , Cumplimiento de la Medicación , Estudios Retrospectivos , Trastornos de la Visión , Pruebas del Campo Visual , Campos VisualesRESUMEN
Protein turnover in cells is regulated by the ATP dependent activity of the Hsp90 chaperone. In concert with accessory proteins, ATP hydrolysis drives the obligate Hsp90 dimer through a cycle between open and closed states that is critical for assisting the folding and stability of hundreds of proteins. Cycling is initiated by ATP binding to the ATPase domain, with the chaperone and the active site gates in the dimer in open states. The chaperone then adopts a short-lived, ATP bound closed state with a closed active site gate. The structural and dynamic changes induced in the ATPase domain and active site gate upon nucleotide binding, and their impact on dimer closing are not well understood. We site-specifically 19F-labeled the ATPase domain at the active site gate to enable benchtop and high field 19F NMR spectroscopic studies. Combined with MD simulations, this allowed accurate characterization of pico- to nanosecond time scale motions of the active site gate, as well as slower micro- to millisecond time scale processes resulting from nucleotide binding. ATP binding induces increased flexibility at one of the hinges of the active site gate, a necessary prelude to release of the second hinge and eventual gate closure in the intact chaperone.
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Adenosina Trifosfato , Proteínas HSP90 de Choque Térmico , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Proteínas HSP90 de Choque Térmico/metabolismo , Espectroscopía de Resonancia Magnética , Unión ProteicaRESUMEN
19F NMR spectroscopy is a powerful tool for the study of the structures, dynamics, and interactions of proteins bearing cysteine residues chemically modified with a trifluoroacetone group (CYF residue). 19F NMR relaxation rates for the fluoromethyl group of CYF residues are sensitive to overall rotational tumbling of proteins, fast rotation about the CF3 methyl axis, and the internal motion of the CYF side-chain. To develop a quantitative understanding of these various motional contributions, we used the model-free approach to extend expressions for 19F- T2 NMR relaxation to include side-chain motions for the CYF residue. We complemented the NMR studies with atomic views of methyl rotation and side-chain motions using molecular dynamics simulations. This combined methodology allows for quantitative separation of the contributions of fast pico- to nanosecond dynamics from micro- to millisecond exchange processes to the 19F line width and highlights the utility of the CYF residue as a sensitive reporter of side-chain environment and dynamics in proteins.
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Acetona/análogos & derivados , Cisteína/química , Simulación de Dinámica Molecular , Acetona/química , Flúor , Espectroscopía de Resonancia MagnéticaRESUMEN
Hsp90 is a crucial chaperone whose ATPase activity is fundamental for stabilizing and activating a diverse array of client proteins. Binding and hydrolysis of ATP by dimeric Hsp90 drive a conformational cycle characterized by fluctuations between a compact, N- and C-terminally dimerized catalytically competent closed state and a less compact open state that is largely C-terminally dimerized. We used 19F and 1H dynamic nuclear magnetic resonance (NMR) spectroscopy to study the opening and closing kinetics of Hsp90 and to determine the kcat for ATP hydrolysis. We derived a set of coupled ordinary differential equations describing the rate laws for the Hsp90 kinetic cycle and used these to analyze the NMR data. We found that the kinetics of closing and opening for the chaperone are slow and that the lower limit for kcat of ATP hydrolysis is â¼1 s-1. Our results show that the chemical step is optimized and that Hsp90 is indeed a "perfect" enzyme.
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Adenosina Trifosfatasas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Pruebas de Enzimas/métodos , Imagen por Resonancia Magnética con Fluor-19 , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Hidrólisis , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutación , Conformación Proteica , Multimerización de Proteína , Espectroscopía de Protones por Resonancia Magnética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.
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Dominio Catalítico , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Análisis Mutacional de ADN , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , UbiquitinaciónRESUMEN
Ring1-YY1-binding protein (RYBP) is a member of the non-canonical polycomb repressive complex 1 (PRC1), and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF) domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs), we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR) repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP) inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding.
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Reparación del ADN/genética , Recombinación Homóloga/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Animales , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Proteínas RepresorasRESUMEN
Cells are exposed to thousands of DNA damage events on a daily basis. This damage must be repaired to preserve genetic information and prevent development of disease. The most deleterious damage is a double-strand break (DSB), which is detected and repaired by mechanisms known as non-homologous end-joining (NHEJ) and homologous recombination (HR), which are components of the DNA damage response system. NHEJ is an error-prone first line of defense, whereas HR invokes error-free repair and is the focus of this review. The functions of the protein components of HR-driven DNA repair are regulated by the coordinated action of post-translational modifications including lysine acetylation, phosphorylation, ubiquitination, and SUMOylation. The latter two mechanisms are fundamental for recognition of DSBs and reorganizing chromatin to facilitate repair. We focus on the structures and molecular mechanisms for the protein components underlying synthesis, recognition, and cleavage of K63-linked ubiquitin chains, which are abundant at damage sites and obligatory for DSB repair. The forward flux of the K63-linked ubiquitination cascade is driven by the combined activity of E1 enzyme, the heterodimeric E2 Mms2-Ubc13, and its cognate E3 ligases RNF8 and RNF168, which is balanced through the binding and cleavage of chains by the deubiquitinase BRCC36, and the proteasome, and through the binding of chains by recognition modules on repair proteins such as RAP80. We highlight a number of aspects regarding our current understanding for the role of kinetics and dynamics in determining the function of the enzymes and chain recognition modules that drive K63 ubiquitination.
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Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Recombinación Homóloga , Lisina/metabolismo , Ubiquitinación , Eucariontes , CinéticaRESUMEN
Isoform 1 of the mammalian Na(+)/H(+) exchanger (NHE1) is a ubiquitously expressed plasma membrane pH regulatory protein. It removes one intracellular H(+) in exchange for one extracellular Na(+). The 500 N-terminal amino acids comprise the catalytic membrane domain and fold into 12 transmembrane (TM) segments. To gain insight into the structure and function of human NHE1, a region spanning transmembrane domains VI and VII was expressed and purified, and the structure was determined using nuclear magnetic resonance (NMR). Segment VI includes two structurally conserved regions corresponding to two short α-helices involving residues 229-236 and 239-247. Segment VII includes one long helical region spanning residues 255-274. The NMR structure of the peptide containing transmembrane domains VI and VII was very similar to the previously published structures of the single-transmembrane segments except that TM VII was not kinked. Tryptophan scanning site-directed mutagenesis of TM VI demonstrated that mutation of residues V240-V245 to tryptophan eliminated NHE1 activity when the full length protein was expressed in cells. In contrast, mutants F246W and E247W were functional. Double mutant V242F/F260V retained activity, while the individual mutations were not active. The results suggest that the region of TM VI from V240 to V245 is closely associated with TM VII and that, in agreement with the NMR structure of VI-VII segments, V242 and F260 are in close association. A study of two transmembrane peptides provides further insight into the structure of the NHE1 protein.
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Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas de Transporte de Catión/genética , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a central role in maintaining cellular homeostasis by facilitating activation of a large number of client proteins. ATP-dependent client activation by Hsp90 is tightly regulated by a host of co-chaperone proteins that control progression through the activation cycle. ATPase stimulation of Hsp90 by Aha1p requires a conserved RKxK motif that interacts with the catalytic loop of Hsp90. In this study, we explore the role of this RKxK motif in the biological and biochemical properties of Hch1p. We found that this motif is required for Hch1p-mediated ATPase stimulation in vitro, but mutations that block stimulation do not impair the action of Hch1p in vivo. This suggests that the biological function of Hch1p is not directly linked to ATPase stimulation. Moreover, a mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p but not by Hch1p. Our work here suggests that both Hch1p and Aha1p regulate Hsp90 function through interaction with the catalytic loop but do so in different ways.
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Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Chaperoninas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Mutación Missense , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Dominio Catalítico , Proteínas HSP90 de Choque Térmico/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
We present the detection of five planets--Kepler-62b, c, d, e, and f--of size 1.31, 0.54, 1.95, 1.61 and 1.41 Earth radii (Râ), orbiting a K2V star at periods of 5.7, 12.4, 18.2, 122.4, and 267.3 days, respectively. The outermost planets, Kepler-62e and -62f, are super-Earth-size (1.25 Râ < planet radius ≤ 2.0 Râ) planets in the habitable zone of their host star, respectively receiving 1.2 ± 0.2 times and 0.41 ± 0.05 times the solar flux at Earth's orbit. Theoretical models of Kepler-62e and -62f for a stellar age of ~7 billion years suggest that both planets could be solid, either with a rocky composition or composed of mostly solid water in their bulk.
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Planetas , Agua , Exobiología , Medio Ambiente Extraterrestre , Modelos Teóricos , Estrellas CelestialesRESUMEN
The NHE1 isoform of the Na(+)/H(+) exchanger is present in the plasma membrane of the mammalian myocardium where it functions to regulate intracellular pH by exchanging one external Na(+) for an internal proton. The protein is involved in myocardial ischemia/reperfusion damage and in heart hypertrophy. Topology models and experimental evidence suggest that of the 815 amino acids of the protein, approximately 500 are embedded or closely associated with the lipid bilayer while the balance form a cytosolic, regulatory carboxyl-terminal tail. The precise structure of NHE1 is not known although that of an Escherichia coli homolog, NhaA, has been determined. The structures of fragments of the NHE1 membrane domain have been examined by nuclear magnetic resonance. Several transmembrane segments have a general structure of an extended central region flanked by helical segments. The extended regions often contain amino acids that are important in protein function and possibly in cation coordination and transport. The E. coli Na(+)/H(+) exchanger NhaA has a novel fold consisting in part of two helical transmembrane segments with interrupted regions crossing amid the lipid bilayer. The similarity between the crystal structure of NhaA and partial structures of NHE1 suggests that there may be similarities in the mechanism of Na(+)/H(+) exchange. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".