RESUMEN
Oxidative and nitrative stress have been implicated in the molecular mechanisms underlying a variety of biological processes and disease states including cancer, aging, cardiovascular disease, neurological disorders, diabetes, and alcohol-induced liver injury. One marker of nitrative stress is the formation of 3-nitrotyrosine, or protein tyrosine nitration (PTN), which has been observed during inflammation and tissue injury; however, the role of PTN in the progression or possibly the pathogenesis of disease is still unclear. We show in a model of alcohol-induced liver injury that an increase in PTN occurs in hepatocyte nuclei within the liver of wild-type male C57BL/6J mice following chronic ethanol exposure (28 days). High-resolution mass spectrometric analysis of isolated hepatic nuclei revealed several novel sites of tyrosine nitration on histone proteins. Histone nitration sites were validated by tandem mass spectrometry (MS/MS) analysis of representative synthetic nitropeptides equivalent in sequence to the respective nitrotyrosine sites identified in vivo. We further investigated the potential structural impact of the novel histone H3 Tyr41 (H3Y41) nitration site identified using molecular dynamics (MD) simulations. MD simulations of the nitrated and non-nitrated forms of histone H3Y41 showed significant structural changes at the DNA interface upon H3Y41 nitration. The results from this study suggest that, in addition to other known post-translational modifications that occur on histone proteins (e.g., acetylation and methylation), PTN could induce chromatin structural changes, possibly affecting gene transcription processes associated with the development of alcohol-induced liver injury.
Asunto(s)
Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Etanol/metabolismo , Histonas/análisis , Nitratos/metabolismo , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Histonas/metabolismo , Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Estrés Oxidativo , Espectrometría de Masas en Tándem , Tirosina/análisis , Tirosina/metabolismoRESUMEN
Malignant transformation of fallopian tube secretory epithelial cells (FTSECs) is a key contributing event to the development of high-grade serous ovarian carcinoma (HGSOC). Our recent findings implicate oncogenic transformative events in chronic iron-exposed FTSECs, including increased expression of oncogenic mediators, increased telomerase transcripts, and increased growth/migratory potential. Herein, we extend these studies by implementing an integrated transcriptomic and mass spectrometry-based proteomics approach to identify global miRNA and protein alterations, for which we also investigate a subset of these targets to iron-induced functional alterations. Proteomic analysis identified > 4500 proteins, of which 243 targets were differentially expressed. Sixty-five differentially expressed miRNAs were identified, of which 35 were associated with the "top" proteomic molecules (> fourfold change) identified by Ingenuity Pathway Analysis. Twenty of these 35 miRNAs are at the 14q32 locus (encoding a cluster of 54 miRNAs) with potential to be regulated by DNA methylation and histone deacetylation. At 14q32, miR-432-5p and miR-127-3p were ~ 100-fold downregulated whereas miR-138-5p was 16-fold downregulated at 3p21 in chronic iron-exposed FTSECs. Combinatorial treatment with methyltransferase and deacetylation inhibitors reversed expression of these miRNAs, suggesting chronic iron exposure alters miRNA expression via epigenetic alterations. In addition, PAX8, an important target in HGSOC and a potential miRNA target (from IPA) was epigenetically deregulated in iron-exposed FTSECs. However, both PAX8 and ALDH1A2 (another IPA-predicted target) were experimentally identified to be independently regulated by these miRNAs although TERT RNA was partially regulated by miR-138-5p. Interestingly, overexpression of miR-432-5p diminished cell numbers induced by long-term iron exposure in FTSECs. Collectively, our global profiling approaches uncovered patterns of miRNA and proteomic alterations that may be regulated by genome-wide epigenetic alterations and contribute to functional alterations induced by chronic iron exposure in FTSECs. This study may provide a platform to identify future biomarkers for early ovarian cancer detection and new targets for therapy.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Trompas Uterinas/efectos de los fármacos , Trompas Uterinas/metabolismo , Compuestos Férricos/farmacología , Sitios Genéticos , MicroARNs/genética , Proteoma/genética , Compuestos de Amonio Cuaternario/farmacología , Transcriptoma/efectos de los fármacos , Azacitidina/farmacología , Biomarcadores de Tumor/genética , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Regulación hacia Abajo/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Proteína del Locus del Complejo MDS1 y EV11/genética , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , MicroARNs/metabolismo , Neoplasias Ováricas/genética , Proteómica/métodos , Transfección , Vorinostat/farmacologíaRESUMEN
We describe a 7-year-old male with high functioning autism spectrum disorder (ASD) and maternally-inherited rare missense variant of Synaptotagmin-like protein 4 (SYTL4) gene (Xq22.1; c.835C>T; p.Arg279Cys) and an unknown missense variant of Transmembrane protein 187 (TMEM187) gene (Xq28; c.708G>T; p. Gln236His). Multiple in-silico predictions described in our study indicate a potentially damaging status for both X-linked genes. Analysis of predicted atomic threading models of the mutant and the native SYTL4 proteins suggest a potential structural change induced by the R279C variant which eliminates the stabilizing Arg279-Asp60 salt bridge in the N-terminal half of the SYTL4, affecting the functionality of the protein's critical RAB-Binding Domain. In the European (Non-Finnish) population, the allele frequency for this variant is 0.00042. The SYTL4 gene is known to directly interact with several members of the RAB family of genes, such as, RAB27A, RAB27B, RAB8A, and RAB3A which are known autism spectrum disorder genes. The SYTL4 gene also directly interacts with three known autism genes: STX1A, SNAP25 and STXBP1. Through a literature-based analytical approach, we identified three of five (60%) autism-associated serum microRNAs (miRs) with high predictive power among the total of 298 mouse Sytl4 associated/predicted microRNA interactions. Five of 13 (38%) miRs were differentially expressed in serum from ASD individuals which were predicted to interact with the mouse equivalent Sytl4 gene. TMEM187 gene, like SYTL4, is a protein-coding gene that belongs to a group of genes which host microRNA genes in their introns or exons. The novel Q236H amino acid variant in the TMEM187 in our patient is near the terminal end region of the protein which is represented by multiple sequence alignments and hidden Markov models, preventing comparative structural analysis of the variant harboring region. Like SYTL4, the TMEM187 gene is expressed in the brain and interacts with four known ASD genes, namely, HCFC1; TMLHE; MECP2; and GPHN. TMM187 is in linkage with MECP2, which is a well-known determinant of brain structure and size and is a well-known autism gene. Other members of the TMEM gene family, TMEM132E and TMEM132D genes are associated with bipolar and panic disorders, respectively, while TMEM231 is a known syndromic autism gene. Together, TMEM187 and SYTL4 genes directly interact with recognized important ASD genes, and their mRNAs are found in extracellular vesicles in the nervous system and stimulate target cells to translate into active protein. Our evidence shows that both these genes should be considered as candidate genes for autism. Additional biological testing is warranted to further determine the pathogenicity of these gene variants in the causation of autism.
Asunto(s)
Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Encéfalo/metabolismo , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , MicroARNs/genética , Mutación Missense/genética , Unión Proteica , Proteínas de Transporte Vesicular/genéticaRESUMEN
The key regulatory enzymes of glycogenolysis are phosphorylase kinase, a hetero-oligomer with four different types of subunits, and glycogen phosphorylase, a homodimer. Both enzymes are activated by phosphorylation and small ligands, and both enzymes have distinct isoforms that are predominantly expressed in muscle, liver, or brain; however, whole-transcriptome high-throughput sequencing analyses show that in brain both of these enzymes are likely composed of subunit isoforms representing all three tissues. This Minireview examines the regulatory properties of the isoforms of these two enzymes expressed in the three tissues, focusing on their potential regulatory similarities and differences. Additionally, the activity, structure, and regulation of the remaining enzyme necessary for glycogenolysis, glycogen-debranching enzyme, are also reviewed.
Asunto(s)
Encéfalo/enzimología , Encéfalo/metabolismo , Glucógeno Fosforilasa/metabolismo , Glucogenólisis , Fosforilasa Quinasa/metabolismo , Animales , Metabolismo Energético , Glucógeno/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/química , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Glucógeno Fosforilasa/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Isoenzimas/metabolismo , Ligandos , Fosforilasa Quinasa/química , Fosforilación , Relación Estructura-Actividad , TranscriptomaRESUMEN
Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αßγδ)4 , and 325 kDa of unique sequence (the mass of an αßγδ protomer). The α, ß and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and ß subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full-length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen-deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N-terminal glycoside hydrolase domain and a large C-terminal importin α/ß-like domain. This structure is similar to our previously published model for the homologous ß subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen-deuterium exchange results obtained for this subunit as part of the (αßγδ)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter-subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low-exchanging region in the C-terminus of α that is known to bind the regulatory domain of the catalytic γ subunit.
Asunto(s)
Fosforilasa Quinasa/química , Subunidades de Proteína/química , Sitio Alostérico , Animales , Dominio Catalítico , Medición de Intercambio de Deuterio , Glucogenólisis , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose-1-phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, ß, γ and δ), making the study of its structure challenging. Using hydrogen-deuterium exchange, we have analyzed the regulatory ß subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the ß subunit within the cryoelectron microscopy envelope of PhK.
Asunto(s)
Fosforilasa Quinasa/química , Subunidades de Proteína/química , Animales , Dominio Catalítico , Microscopía por Crioelectrón , Glucogenólisis , Humanos , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: (1) hydrogen/deuterium exchange (HDX), (2) limited proteolysis, and (3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography.
Asunto(s)
Biología Computacional/métodos , Minería de Datos/métodos , Bases de Datos de Proteínas , Espectrometría de Masas/métodos , Proteínas/análisis , Proteoma , Proteómica/métodos , Algoritmos , Animales , Reactivos de Enlaces Cruzados/química , Medición de Intercambio de Deuterio , Ensayos Analíticos de Alto Rendimiento , Humanos , Conformación Proteica , Proteolisis , Reproducibilidad de los Resultados , Programas Informáticos , Flujo de TrabajoRESUMEN
In the six decades since its discovery, phosphorylase kinase (PhK) from rabbit skeletal muscle has usually been studied at 30 °C; in fact, not a single study has examined functions of PhK at a rabbit's body temperature, which is nearly 10 °C greater. Thus, we have examined aspects of the activity, regulation, and structure of PhK at temperatures between 0 and 40 °C. Between 0 and 30 °C, the activity at pH 6.8 of nonphosphorylated PhK predictably increased; however, between 30 and 40 °C, there was a dramatic jump in its activity, resulting in the nonactivated enzyme having a far greater activity at body temperature than was previously realized. This anomalous change in properties between 30 and 40 °C was observed for multiple functions, and both stimulation (by ADP and phosphorylation) and inhibition (by orthophosphate) were considerably less pronounced at 40 °C than at 30 °C. In general, the allosteric control of PhK's activity is definitely more subtle at body temperature. Changes in behavior related to activity at 40 °C and its control can be explained by the near disappearance of hysteresis at physiological temperature. In important ways, the picture of PhK that has emerged from six decades of study at temperatures of ≤30 °C does not coincide with that of the enzyme studied at physiological temperature. The probable underlying mechanism for the dramatic increase in PhK's activity between 30 and 40 °C is an abrupt change in the conformations of the regulatory ß and catalytic γ subunits between these two temperatures.
Asunto(s)
Temperatura Corporal , Fosforilasa Quinasa/metabolismo , Animales , Activación Enzimática , Femenino , Fosforilación , ConejosRESUMEN
Phosphorylase kinase (PhK) is a 1.3 MDa (αßγδ)4 enzyme complex, in which αßγδ protomers associate in D2 symmetry to form two large octameric lobes that are interconnected by four bridges. The approximate locations of the subunits have been mapped in low-resolution cryo-electron microscopy structures of the complex; however, the disposition of the subunits within the complex remains largely unknown. We have used partial proteolysis and chemical footprinting in combination with high-resolution mass spectrometry to identify surface-exposed regions of the intact nonactivated and phospho-activated conformers. In addition to the known interaction of the γ subunit's C-terminal regulatory domain with the δ subunit (calmodulin), our exposure results indicate that the catalytic core of γ may also anchor to the PhK complex at the bottom backside of its C-terminal lobe facing away from the active site cleft. Exposed loops on the α and ß regulatory subunits within the complex occur at regions overlapping with tissue-specific alternative RNA splice sites and regulatory phosphorylatable domains. Their phosphorylation alters the surface exposure of α and ß, corroborating previous biophysical and biochemical studies that detected phosphorylation-dependent conformational changes in these subunits; however, for the first time, specific affected regions have been identified.
Asunto(s)
Fosforilasa Quinasa/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Espectrometría de Masas , Modelos Moleculares , Mapeo Peptídico , Fosforilasa Quinasa/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Proteolisis , ConejosRESUMEN
Phosphorylase kinase (PhK) is a hexadecameric (αßγδ)(4) enzyme complex that upon activation by phosphorylation stimulates glycogenolysis. Due to its large size (1.3 MDa), elucidating the structural changes associated with the activation of PhK has been challenging, although phosphoactivation has been linked with an increased tendency of the enzyme's regulatory ß-subunits to self-associate. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of ß on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form ß-ß dimers in the nonactivated PhK complex. This peptide stimulated ß-ß dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of ß competes with its counterpart region in the nonactivated enzyme complex in binding to the catalytic γ-subunit, we were able to formulate a structural model for the phosphoactivation of PhK. In this model, the nonactivated state of PhK is maintained by the interaction between the nonphosphorylated N-termini of ß and the regulatory C-terminal domains of the γ-subunits; phosphorylation of ß weakens this interaction, leading to activation of the γ-subunits.
Asunto(s)
Materiales Biomiméticos/química , Péptidos/química , Fosforilasa Quinasa/química , Fosforilasa Quinasa/metabolismo , Sitios de Unión , Dominio Catalítico , Activación Enzimática , Glucogenólisis , Modelos Moleculares , Complejos Multienzimáticos/química , Oxidación-Reducción , Fosforilación , Estructura Secundaria de ProteínaRESUMEN
For over four decades free Mg(2+) ions, that is, those in excess of MgATP, have been reported to affect a wide variety of properties of phosphorylase kinase (PhK), including its affinity for other molecules, proteolysis, chemical crosslinking, phosphorylation, binding to certain monoclonal antibodies, and activity, which is stimulated. Additionally, for over three decades Mg(2+) has been known to act synergistically with Ca(2+) , another divalent activator of PhK, to affect even more properties of the enzyme. During all of this time, however, no study has been performed to determine the overall effects of free Mg(2+) ions on the physical properties of PhK, even though the effects of Ca(2+) ions on PhK's properties are well documented. In this study, changes in the physicochemical properties of PhK induced by Mg(2+) under nonactivating (pH 6.8) and activating (pH 8.2) conditions were investigated by circular dichroism spectroscopy, zeta potential analyses, dynamic light scattering, second derivative UV absorption, negative stain electron microscopy, and differential chemical crosslinking. The effects of the activator Mg(2+) on some of the properties of PhK measured by these techniques were found to be quite different at the two pH values, and displayed both differences and similarities with the effects previously reported to be induced by the activator Ca(2+) (Liu et al., Protein Sci 2008;17:2111-2119). The similarities may reflect the fact that both cations are activators, and foremost among their similarities is the dramatically less negative zeta potential induced by their binding to PhK.
Asunto(s)
Magnesio/química , Magnesio/metabolismo , Fosforilasa Quinasa/química , Fosforilasa Quinasa/metabolismo , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/metabolismo , Cationes/química , Cationes/metabolismo , Dicroismo Circular , Dinitrofluorobenceno/análogos & derivados , Dinitrofluorobenceno/química , Dinitrofluorobenceno/metabolismo , Luz , Conformación Proteica , Dispersión de Radiación , Electricidad EstáticaRESUMEN
Phosphorylase kinase (PhK) is a hexadecameric (αßγδ)(4) complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, ß, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αßγδ)(2) lobes joined with D(2) symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the ß subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the ß subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of ß was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αßγδ)(4) complex. An atomic model displaying tertiary structure for the entire ß subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the ß subunits, they were directly determined to compose the four interconnecting bridges in the (αßγδ)(4) kinase core, because a ß(4) subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the ß subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.
Asunto(s)
Fosforilasa Quinasa/química , Subunidades de Proteína/química , Secuencia de Aminoácidos , Animales , Reactivos de Enlaces Cruzados/química , Dinitrofluorobenceno/análogos & derivados , Dinitrofluorobenceno/química , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Conejos , Espectrometría de Masas en TándemRESUMEN
Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, ß, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the ß subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple ß subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αßγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.
Asunto(s)
Dominio Catalítico , Músculo Esquelético/enzimología , Fosforilasa Quinasa , Subunidades de Proteína/análisis , Catálisis , Espectrometría de Masas , Músculo Esquelético/metabolismo , Fosforilasa Quinasa/análisis , Fosforilasa Quinasa/química , Fosforilasa Quinasa/metabolismo , Fosforilación , Conformación Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína/químicaRESUMEN
This chapter explores the structural responses of a massive, hetero-oligomeric protein complex to a single allosteric activator as probed by a wide range of chemical, biochemical, and biophysical approaches. Some of the approaches used are amenable only to large protein targets, whereas others push the limits of their utility. Some of the techniques focus on individual subunits, or portions thereof, while others examine the complex as a whole. Despite the absence of crystallographic data for the complex, the diverse techniques identify and implicate a small region of its catalytic subunit as the master allosteric activation switch for the entire complex.
Asunto(s)
Proteínas/química , Regulación Alostérica/fisiología , Calcio/farmacología , Transferencia Resonante de Energía de Fluorescencia , Inmunoquímica , Microscopía Electrónica , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína , Proteínas/metabolismo , Proteínas/ultraestructuraRESUMEN
Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, stimulates energy production from glycogen in the cascade activation of glycogenolysis. Its large homologous alpha and beta subunits regulate the activity of the catalytic gamma subunit and account for 81% of PhK's mass. Both subunits are thought to be multidomain structures, and recent predictions based on their sequences suggest the presence of potentially functional glucoamylase (GH15)-like domains near their amino termini. We present the first experimental evidence of such a domain in PhK by demonstrating that the glucoamylase inhibitor acarbose binds PhK, perturbs its structure, and stimulates its kinase activity.
Asunto(s)
Acarbosa/farmacología , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , Fosforilasa Quinasa/química , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos , Humanos , Hipoglucemiantes , Fosforilasa Quinasa/efectos de los fármacos , Unión Proteica , Conformación ProteicaRESUMEN
Understanding the regulatory interactions among the 16 subunits of the (alphabetagammadelta)(4) phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells alpha, beta, gamma, and delta subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding alphagammadelta trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries. The catalytic activity of rPhK at pH 8.2 and its ratio of activities at pH 6.8 versus pH 8.2 were comparable to those of PhK purified from rabbit muscle (RM PhK), as was the hysteresis (autoactivation) in the rate of product formation at pH 6.8. Both the rPhK and alphagammadelta exhibited only a very low Ca(2+)-independent activity and a Ca(2+)-dependent activity similar to that of the native holoenzyme with [Ca(2+)](0.5) of 0.4 microM for the RM PhK, 0.7 microM for the rPhK, and 1.5 microM for the alphagammadelta trimer. The RM PhK, rPhK, and alphagammadelta subcomplex were also all activated through self-phosphorylation. Using cross-linking and limited proteolysis, the alpha-gamma intersubunit contacts previously observed within the intact RM PhK complex were also observed within the recombinant alphagammadelta subcomplex. Our results indicate that both the rPhK and alphagammadelta subcomplex are promising models for future structure-function studies on the regulation of PhK activity through intersubunit contacts, because both retained the regulatory properties of the enzyme purified from skeletal muscle.
Asunto(s)
Músculo Esquelético/enzimología , Fosforilasa Quinasa/metabolismo , Subunidades de Proteína/metabolismo , Animales , Baculoviridae/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Cinética , Modelos Animales , Músculo Esquelético/metabolismo , Fosforilación , Subunidades de Proteína/química , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC(50)(Ca(2+)) values for calmodulin binding and enzyme activation from the control values of 182 +/- 2 and 422 +/- 22 nm to 116 +/- 2 and 300 +/- 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557-7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC(50)(Ca(2+)) values for calmodulin binding and enzyme activation to 77 +/- 2 and 130 +/- 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca(2+) concentration of 50-100 nm.
Asunto(s)
Calcio/química , Calmodulina/química , Óxido Nítrico Sintasa de Tipo III/química , Proteínas Proto-Oncogénicas c-akt/química , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Bovinos , Reductasas del Citocromo/química , Reductasas del Citocromo/genética , Reductasas del Citocromo/metabolismo , Humanos , Mutación Missense , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación/fisiología , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estrés Fisiológico/fisiologíaRESUMEN
Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Fosforilasa Quinasa/metabolismo , Subunidades de Proteína/metabolismo , Animales , Calcio/farmacología , Reactivos de Enlaces Cruzados/farmacología , Enzimas Inmovilizadas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Fosforilasa Quinasa/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Conejos , Succinimidas/farmacología , Extractos de TejidosRESUMEN
Skeletal muscle phosphorylase kinase (PhK) is a Ca(2+)-dependent enzyme complex, (alpha beta gamma delta)(4), with the delta subunit being tightly bound endogenous calmodulin (CaM). The Ca(2+)-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the "excitation-contraction-energy production triad." Although the Ca(2+)-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic gamma and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the gamma subunit sharing high sequence similarity with the Ca(2+)-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca(2+) stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca(2+) of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca(2+)-binding proteins troponin C and CaM.
Asunto(s)
Metabolismo Energético , Contracción Muscular , Músculo Esquelético/fisiología , Fosforilasa Quinasa/química , Animales , Calcio/fisiología , Calmodulina/química , Calmodulina/fisiología , Dominio Catalítico , Cromatografía Liquida , Reactivos de Enlaces Cruzados/química , Dimerización , Humanos , Fosforilasa Quinasa/fisiología , Espectrometría de Masa por Ionización de Electrospray , Troponina C/fisiología , Troponina I/fisiologíaRESUMEN
Chemical cross-linking and high resolution MS have been integrated successfully to capture protein interactions and provide low resolution structural data for proteins that are refractive to analyses by NMR or crystallography. Despite the versatility of these combined techniques, the array of products that is generated from the cross-linking and proteolytic digestion of proteins is immense and generally requires the use of labeling strategies and/or data base search algorithms to distinguish actual cross-linked peptides from the many side products of cross-linking. Most strategies reported to date have focused on the analysis of small cross-linked protein complexes (<60 kDa) because the number of potential forms of covalently modified peptides increases dramatically with the number of peptides generated from the digestion of such complexes. We report herein the development of a user-friendly search engine, CrossSearch, that provides the foundation for an overarching strategy to detect cross-linked peptides from the digests of large (>or=170-kDa) cross-linked proteins, i.e. conjugates. Our strategy combines the use of a low excess of cross-linker, data base searching, and Fourier transform ion cyclotron resonance MS to experimentally minimize and theoretically cull the side products of cross-linking. Using this strategy, the (alpha beta gamma delta)(4) phosphorylase kinase model complex was cross-linked to form with high specificity a 170-kDa betagamma conjugate in which we identified residues involved in the intramolecular cross-linking of the 125-kDa beta subunit between its regulatory N terminus and its C terminus. This finding provides an explanation for previously published homodimeric two-hybrid interactions of the beta subunit and suggests a dynamic structural role for the regulatory N terminus of that subunit. The results offer proof of concept for the CrossSearch strategy for analyzing conjugates and are the first to reveal a tertiary structural element of either homologous alpha or beta regulatory subunit of phosphorylase kinase.