RESUMEN
Cilia are organelles specialized in movement and signal transduction. The ciliary transient receptor potential ion channel polycystin-2 (TRPP2) controls elementary cilia-mediated physiological functions ranging from male fertility and kidney development to left-right patterning. However, the molecular components translating TRPP2 channel-mediated Ca2+ signals into respective physiological functions are unknown. Here, we show that the Ca2+-regulated mitochondrial ATP-Mg/Pi solute carrier 25 A 25 (SLC25A25) acts downstream of TRPP2 in an evolutionarily conserved metabolic signaling pathway. We identify SLC25A25 as an essential component in this cilia-dependent pathway using a genome-wide forward genetic screen in Drosophila melanogaster, followed by a targeted analysis of SLC25A25 function in zebrafish left-right patterning. Our data suggest that TRPP2 ion channels regulate mitochondrial SLC25A25 transporters via Ca2+ establishing an evolutionarily conserved molecular link between ciliary signaling and mitochondrial metabolism.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Cilios/metabolismo , Canales Catiónicos TRPP/metabolismo , Animales , Antiportadores/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Drosophila melanogaster/metabolismo , Heterocigoto , Humanos , Canales Iónicos/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Transducción de Señal , Pez CebraRESUMEN
Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17ß-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer-monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1-MRPP2-MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/química , Metiltransferasas/química , Modelos Moleculares , Complejos Multienzimáticos/química , ARN Mitocondrial/química , ARN de Transferencia/química , Ribonucleasa P/química , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Cristalografía por Rayos X , Humanos , Metiltransferasas/metabolismo , Complejos Multienzimáticos/metabolismo , ARN Mitocondrial/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P/metabolismo , Dispersión del Ángulo PequeñoRESUMEN
Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants.
Asunto(s)
Galactosemias/genética , Relación Estructura-Actividad , Factores Complejos Ternarios/química , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , Sitios de Unión/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Galactosa/química , Galactosa/metabolismo , Galactosemias/metabolismo , Galactosemias/patología , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Factores Complejos Ternarios/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/químicaRESUMEN
Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Transporte de Membrana Mitocondrial/química , Vitamina B 12/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intracelular , Enfermedades Metabólicas/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial/genética , Chaperonas Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Nitrorreductasas/química , Oxidorreductasas , Fenotipo , Unión Proteica , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Hand trauma is a top presenting complaint to hospital emergency departments (EDs) and can become costly if not treated effectively. The cornerstone for initial management of the traumatized hand is application of a splint. Improving splinting practice could potentially produce tangible benefits in terms of quality of care and costs to society. OBJECTIVES: We sought to determine the following: 1) whether the present standard of ED splinting was appropriate and 2) whether a strategically planned educational intervention could improve the existing care. METHODS: We used a pre- and postprospective educational intervention study design. In the preintervention phase, patients referred to our hand clinic were assessed for injury and splint type. Splinting appropriateness was evaluated according to a predetermined hand surgeons' expert consensus. Next, an educational intervention was targeted at all ED staff at our institution. Postintervention, all patients were again evaluated for splint appropriateness. A follow-up evaluation was performed at 1 year to see the long-term effects of the intervention. RESULTS: The most common mechanism of injury of referred patients was falling (35%), and the most frequent injury was metacarpal fracture (40%). Splint appropriateness increased significantly postintervention from 49% to 69% (p = 0.048). At follow-up after 1 year, splinting appropriateness was 70% (p = 0.041). CONCLUSION: Appropriate hand splinting practice is essential for hand trauma management. Our results show that an educational intervention can successfully improve splinting practice. This quality of care initiative was low-cost and demonstrated persistence at 1 year of follow-up.
Asunto(s)
Medicina de Emergencia/educación , Medicina de Emergencia/normas , Traumatismos de la Mano/terapia , Capacitación en Servicio , Férulas (Fijadores)/normas , Adulto , Femenino , Estudios de Seguimiento , Humanos , Análisis de Series de Tiempo Interrumpido , Masculino , Persona de Mediana Edad , Mejoramiento de la Calidad , Factores de Tiempo , Adulto JovenRESUMEN
Cystathionine ß-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe(443), Asp(444), Gln(445), and Asp(538)) and for AdoMet-driven inter-domain communication (Phe(443), Asp(538)). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis.
Asunto(s)
Cistationina betasintasa/química , S-Adenosilmetionina/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Calcium kidney stones are common and recurrences are often not preventable by available empiric remedies. Their etiology is multifactorial and polygenic, and an increasing number of genes are implicated. Their identification will enable improved management. METHODS: DNA from three stone-formers in a Southampton family (UK) and two from an Italian family were analyzed independently by whole exome sequencing and selected variants were genotyped across all available members of both pedigrees. A disease variant of SLC25A25 (OMIM 608745), encoding the mitochondrial ATP-Mg/Pi carrier 3 (APC3) was identified, and analyzed structurally and functionally with respect to its calcium-regulated transport activity. RESULTS: All five patients had a heterozygous dominant SLC25A25 variant (rs140777921; GRCh37.p13: chr 9 130868670 G>C; p.Gln349His; Reference Sequence NM_001006641.3). Non-stone formers also carried the variant indicating incomplete penetrance. Modeling suggests that the variant lacks a conserved polar interaction, which may cause structural instability. Calcium-regulated ATP transport was reduced to ~20% of the wild type, showing a large reduction in function. CONCLUSION: The transporter is important in regulating mitochondrial ATP production. This rare variant may increase urine lithogenicity through impaired provision of ATP for solute transport processes in the kidney, and/or for purinergic signaling. Variants found in other genes may compound this abnormality.
Asunto(s)
Proteínas de Unión al Calcio/genética , Secuenciación del Exoma , Genes Mitocondriales , Variación Genética , Cálculos Renales/diagnóstico , Cálculos Renales/etiología , Proteínas de Transporte de Membrana Mitocondrial/genética , Adolescente , Adulto , Anciano , Alelos , Secuencia de Aminoácidos , Bancos de Muestras Biológicas , Biomarcadores , Familia , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Cálculos Renales/metabolismo , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple , Conformación Proteica , Relación Estructura-Actividad , Evaluación de Síntomas , Reino Unido , Adulto JovenRESUMEN
The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.
Asunto(s)
Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/metabolismo , Fenilalanina/metabolismo , Regulación Alostérica , Sitios de Unión , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Dispersión del Ángulo PequeñoRESUMEN
Interactive liquid chromatography (iLC) for polymer analysis is usually applied to the characterisation of distributions other than molar mass. In particular, its use for the determination of chemical-composition, functionality-type and tacticity distributions has been demonstrated. The application of iLC for the determination of molar mass distributions (MMDs), however, has not yet been fully explored. An expanded version of the reversed-phase liquid chromatography model has been developed to describe and predict how the retention behaviour of polydisperse polystyrene samples changes with molar mass. The relationship between molar mass and the parameters of the model has been investigated in some detail and non-linear correlations were found. From the model and the relationships between the model parameters and molar mass, calibration curves (retention time versus molar mass) were constructed to predict changes in chromatographic selectivity across a given molar mass range. These calibration curves were compared to experimentally obtained curves and, in most cases, excellent agreement was found. The dramatic enhancement in selectivity that can be obtained with iLC in comparison to size-exclusion chromatography (SEC) was illustrated by measuring matrix-assisted laser desorption ionisation (MALDI) MS spectra of fractions collected during a gradient-LC separation. In the low-molar mass range, essentially monodisperse fractions were obtained. Calibration curves, predicted by the model and validated experimentally using narrow-dispersity standards and MALDI-MS spectra of fractions, were used to determine the molar mass distribution of some narrowly distributed polystyrene samples. Molar mass distributions for such standards were found to be somewhat lower than the values reported by the manufacturers. The results also deviated from those obtained by MALDI-MS.
Asunto(s)
Peso Molecular , Calibración , Cromatografía en Gel/métodos , Modelos Teóricos , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In this paper we describe how the existing theories to describe retention and peak width in isocratic and gradient-elution liquid chromatography can be expanded to describe the retention behaviour of natural and synthetic repetitive polymers, which feature distributions of molecules with different masses (and often different structures) rather than unambiguous molecular formulas. For polydisperse samples, it is vital that the model accommodates (isocratic) elution of sample components before the onset of a gradient, elution during the gradient, and elution after the completion of the gradient. The expanded models can readily be implemented in standard spreadsheet software, such as Excel. We have created such spreadsheets based on the conventional model for retention in reversed-phase liquid chromatography (RPLC) and on two different models for retention in normal-phase liquid chromatography. The implementation allows an easy visualization of the theoretical concept. Up to three different polymeric series can be entered, with a total of up to 100 peaks being computed and displayed in isocratic or gradient-elution chromatograms. Also visualized are "retention models" (diagrams of isocratic retention vs. composition) and "calibration curves" (retention or elution composition vs. molecular mass or degree of polymerization). The coefficients in the isocratic retention model may be correlated, as has often been observed in RPLC. It is shown that under certain conditions such a correlation corresponds to the existence of so-called critical (isocratic) conditions, at which all the members of a given polymeric series (same composition and end groups, different number of repeat units) show co-elution.
Asunto(s)
Cromatografía Liquida/métodos , Polímeros/química , Simulación por ComputadorRESUMEN
This paper illustrates how conventional models of chromatographic behaviour can be used to predict the separation behaviour of polydisperse macromolecules. Using polystyrene and polymethylmethacrylate homo- and co-polymeric standards, the models were validated by comparing experimental retention behaviour with that predicted by the chromatographic model. The experimental retention time of each of the samples was entered into a spreadsheet application, which calculated the parameters that best described retention (for a given model). When a correlation between the relevant parameters and molecular mass was established, that correlation was used to predict the change in retention behaviour over the molecular-mass range. An expression introduced in a previous paper, to calculate the critical mobile-phase composition of a homopolymer was validated using polystyrene homopolymers. A second expression, which can predict the elution behaviour of copolymers, was also validated. This expression can predict the retention of a copolymer, based solely onthe retention of the homopolymeric units that make up the copolymer.
Asunto(s)
Cromatografía Liquida/métodos , Calibración , Modelos QuímicosRESUMEN
This study illustrates how retention models can be used to accurately predict the retention behaviour of polydisperse macromolecules in LC separations. It highlights that the number of experiments required can be drastically reduced when the relationship between the model parameters and molecular structure parameters (e.g. molar mass) can be incorporated into one global model. A practical implication of this work is that an appropriate model can then be used for the determination of molar-mass distributions for polydisperse samples. The globalised model can predict retention time as a function of molar mass and gradient slope. Both the original and globalised versions of the model were rigorously validated in terms of the difference between the predicted and experimental retention times. The original model had very low residuals and there was no apparent dependence of the errors on the applied gradient, the molar mass or the retention times. Confidence intervals for the model parameters (S and ln k0) were determined using a bootstrapping analysis of the residual errors in the predicted retention times. Confidence intervals were seen to broaden significantly as the mass of the polymer increased. The parameters were also seen to be highly correlated. For the global model, retention-time residuals remained quite low, even when the number of experiments used to determine the model parameters was reduced from approximately 100 to 10.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Modelos Químicos , Peso MolecularRESUMEN
A new method to eliminate the background spectrum (EBS) during analyte elution in column liquid chromatography (LC) coupled to spectroscopic techniques is proposed. This method takes into account the shape and also intensity differences of the background eluent spectrum. This allows the EBS method to make a better estimation of the background eluent spectrum during analyte elution. This is an advantage for quantification as well as for identification of analytes. The EBS method uses a two-step procedure. First, the baseline spectra are modeled using a limited number of principal components (PCs). Subsequently, an asymmetric least squares (asLS) regression method is applied using these principal components to correct the measured spectra during elution for the background contribution. The asymmetric least squares regression needs one parameter, the asymmetry factor p. This asymmetry factor determines relative weight of positive and negative residuals. Simulations are performed to test the EBS method in well-defined situations. The effect of spectral noise on the performance and the sensitivity of the EBS method for the value of the asymmetry factorp is tested. Two applications of the EBS method are discussed. In the first application, the goal is to extract the analyte spectrum from an LC-Raman analysis. In this case, the EBS method facilitates easy identification of unknown analytes using spectral libraries. In a second application, the EBS method is used for baseline correction in LC-diode array detection (DAD) analysis of polymeric standards during a gradient elution separation. It is shown that the EBS method yields a good baseline correction, without the need to perform a blank chromatographic run.
Asunto(s)
Espectrofotometría Infrarroja/métodos , Espectrofotometría Ultravioleta/métodos , Espectrometría Raman/métodosRESUMEN
A perfluorinated co-polyether was characterised in terms of the number and type of functional end groups present on the molecule. The polymer was separated chromatographically according to the polarity of the polymer end groups and the separation was coupled on-line to an electrospray ionisation time-of-flight mass spectrometer. Negative-mode electrospray ionisation of the relatively non-polar polymer was achieved by post-column addition of a polar constituent to the mobile phase. LC-MS analysis of polydisperse analytes is a highly data intensive technique and manual interpretation of the resulting data can be extremely complicated, especially for the characterisation of copolymers or polymers with end-group distributions. In order to overcome this problem, an automated data-analysis program was developed that allows the user to quickly determine the probability of the presence of a certain molecular compound. The program evaluated data in terms of the possible combinations of monomeric units and end groups that could be combined to make up the mass values present in the mass spectra. Using the program, the polymer can be characterised according to its molar-mass, chemical-composition and functionality-type distributions. A graphical representation of the LC-MS analyses is presented to give a clear overview of the two-dimensional separation. The identification of various end groups on the polymer is also presented graphically, as (a) a histogram (frequency of matches versus time), (b) a two-dimensional plot (masses that match the particular end group combination versus LC retention time) and (c) a plot of average chemical composition versus LC retention time.
Asunto(s)
Cromatografía Liquida/métodos , Flúor/química , Polímeros/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Automatización , Interpretación Estadística de DatosRESUMEN
The substituent patterns of some chemically modified celluloses were characterized as a function of their size distribution, using size-exclusion chromatography coupled to both nuclear magnetic resonance spectroscopy (NMR) and cloud-point measurements. Intact and enzymatically hydrolyzed methyl cellulose (MC) was fractionated according to size, and the level of substitution of the fractions was measured off-line using NMR. Clouding behavior was also measured as a function of size. Clear differences between hydrolyzed and nonhydrolyzed samples were observed using both techniques. For samples that had been selectively hydrolyzed using cellulose-degrading enzymes, NMR data showed a direct link between the degree of degradation and the level of substitution. Differences in the clouding behavior highlighted changes in substituent levels and substituent patterns across the size distribution. The techniques gave valuable and somewhat complementary information on the substituent distributions of the samples before and after enzymatic hydrolysis.