RESUMEN
Starch accumulation in plant tissues provides an important carbon source at night and for regrowth after periods of dormancy and in times of stress. Both É- and ß-amylases (AMYs and BAMs, respectively) catalyze starch hydrolysis, but their functional roles are unclear. Moreover, the presence of catalytically inactive amylases that show starch excess phenotypes when deleted presents an interesting series of questions on how starch degradation is regulated. Plants lacking one of these catalytically inactive ß-amylases, BAM9, were shown to have enhanced starch accumulation when combined with mutations in BAM1 and BAM3, the primary starch degrading BAMs in response to stress and at night, respectively. Importantly, BAM9 has been reported to be transcriptionally induced by stress through activation of SnRK1. Using yeast two-hybrid experiments, we identified the plastid-localized AMY3 as a potential interaction partner for BAM9. We found that BAM9 interacted with AMY3 in vitro and that BAM9 enhances AMY3 activity 3-fold. Modeling of the AMY3-BAM9 complex revealed a previously undescribed N-terminal structural feature in AMY3 that we call the alpha-alpha hairpin that could serve as a potential interaction site. Additionally, AMY3 lacking the alpha-alpha hairpin is unaffected by BAM9. Structural analysis of AMY3 showed that it can form a homodimer in solution and that BAM9 appears to replace one of the AMY3 monomers to form a heterodimer. Collectively these data suggest that BAM9 is a pseudoamylase that activates AMY3 in response to cellular stress, possibly facilitating starch degradation to provide an additional energy source for stress recovery.
RESUMEN
The process for and regulatory mechanism controlling the synthesis and degradation of the polysaccharide starch are only superficially understood. ß-amylases (BAMs) are enzymes that hydrolyze starch into maltose which is further used to drive metabolism and other cellular processes. Most BAMs in plants can function as monomeric enzymes and have hyperbolic kinetics. BAM2 from Arabidopsis thaliana is unusual as it forms a homotetramer, displays sigmoidal kinetics, and is stimulated by the presence of potassium cations (K + ). We used circular dichroism spectroscopy, small-angle X-Ray scattering, and molecular dynamics to investigate the effect of K + on the structure of BAM2 and found that K + induces the formation of an active conformation of BAM2 thereby increasing its activity.
RESUMEN
Malate dehydrogenase (MDH) enzymes catalyze the reversible oxidoreduction of malate to oxaloacetate using NAD(P) as a cofactor. This reaction is vital for metabolism and the exchange of reducing equivalents between cellular compartments. There are more than 100 structures of MDH in the Protein Data Bank, representing species from archaea, bacteria, and eukaryotes. This conserved family of enzymes shares a common nucleotide-binding domain, substrate-binding domain, and subunits associate to form a dimeric or a tetrameric enzyme. Despite the variety of crystallization conditions and ligands in the experimental structures, the conformation and configuration of MDH are similar. The quaternary structure and active site dynamics account for most conformational differences in the experimental MDH structures. Oligomerization appears essential for activity despite each subunit having a structurally independent active site. There are two dynamic regions within the active site that influence substrate binding and possibly catalysis, with one of these regions adjoining the subunit interface. In this review, we introduce the reader to the general structural framework of MDH highlighting the conservation of certain features and pointing out unique differences that regulate MDH enzyme activity.
RESUMEN
Malate dehydrogenase (MDH) catalyzes the interconversion of oxaloacetate and malate coupled to the oxidation/reduction of coenzymes NAD(P)H/NAD(P)+. While most animals have two isoforms of MDH located in the cytosol and mitochondria, all major groups of land plants have at least six MDHs localized to the cytosol, mitochondria, plastids, and peroxisomes. This family of enzymes participates in important reactions in plant cells including photosynthesis, photorespiration, lipid metabolism, and NH4+ metabolism. MDH also helps to regulate the energy balance in the cell and may help the plant cope with various environmental stresses. Despite their functional diversity, all of the plant MDH enzymes share a similar structural fold and act as dimers. In this review, we will introduce readers to our current understanding of the plant MDHs, including their evolution, structure, and function. The focus will be on the MDH enzymes of the model plant Arabidopsis thaliana.
RESUMEN
Starch accumulates in the plastids of green plant tissues during the day to provide carbon for metabolism at night. Starch hydrolysis is catalyzed by members of the ß-amylase (BAM) family, which in Arabidopsis thaliana (At) includes nine structurally and functionally diverse members. One of these enzymes, AtBAM2, is a plastid-localized enzyme that is unique among characterized ß-amylases since it is tetrameric and exhibits sigmoidal kinetics. Sequence alignments show that the BAM domains of AtBAM7, a catalytically inactive, nuclear-localized transcription factor with an N-terminal DNA-binding domain, and AtBAM2 are more closely related to each other than they are to any other AtBAM. Since the BAM2 gene is found in more ancient lineages, it was hypothesized that the BAM7 gene evolved from BAM2. However, analysis of the genomes of 48 flowering plants revealed 12 species that appear to possess a BAM7 gene but lack a BAM2 gene. Upon closer inspection, these BAM7 proteins have a greater percent identity to AtBAM2 than to AtBAM7, and they share all of the AtBAM2 functional residues that BAM7 proteins normally lack. It is hypothesized that these genes may encode BAM2-like proteins although they are currently annotated as BAM7-like genes. To test this hypothesis, a cDNA for the short form of corn BAM7 (ZmBAM7-S) was designed for expression in Escherichia coli. Small-angle X-ray scattering data indicate that ZmBAM7-S has a tetrameric solution structure that is more similar to that of AtBAM2 than to that of AtBAM1. In addition, partially purified ZmBAM7-S is catalytically active and exhibits sigmoidal kinetics. Together, these data suggest that some BAM7 genes may encode a functional BAM2. Exploring and understanding the ß-amylase gene structure could have an impact on the current annotation of genes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , beta-Amilasa , Proteínas de Arabidopsis/química , Catálisis , Proteínas Serina-Treonina Quinasas , Almidón/metabolismo , Zea mays/genética , Zea mays/metabolismo , beta-Amilasa/químicaRESUMEN
The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 25 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle X-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina-specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.
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Amaurosis Congénita de Leber , Factores de Transcripción , Animales , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Amaurosis Congénita de Leber/genética , Dispersión del Ángulo Pequeño , Factores de Transcripción/genética , Difracción de Rayos XRESUMEN
Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. ß-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.
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Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Proteínas Serina-Treonina Quinasas/química , Almidón/metabolismo , beta-Amilasa/química , Secuencia de Aminoácidos , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Dispersión de Radiación , Alineación de Secuencia , Rayos XRESUMEN
Human tetherin, also known as BST-2 or CD317, is a dimeric, extracellular membrane-bound protein that consists of N and C terminal membrane anchors connected by an extracellular domain. BST-2 is involved in binding enveloped viruses, such as HIV, and inhibiting viral release in addition to a role in NF-kB signaling. Viral tethering by tetherin can be disrupted by the interaction with Vpu in HIV-1 in addition to other viral proteins. The structural mechanism of tetherin function is not clear and the effects of human tetherin mutations identified by sequencing consortiums are not known. To address this gap in the knowledge, we used data from the Ensembl database to construct and model known human missense mutations within the ectodomain to investigate how the structure of the ectodomain influences function. From the data, we identified an island of sequence stability within the ectodomain, which corresponds to a functionally and structurally important region identified in previous biochemical and biophysical studies. Most of the modeled mutations had little effect on the structure of the dimer and the coiled-coil, suggesting that the coiled-coil compensates for changes in primary structure. Thus, many of the functional defects observed in previous studies may not be due to changes in tetherin structure, but rather, due to in changes in protein-protein interactions or in aspects of tetherin not currently understood. The lack of structural effects by mutations known to decrease function further illustrates the need for more study of the structure-function connection for this system. Finally, apparent flexibility in tetherin sequence may allow for greater anti-viral activities with a larger number of viruses by reducing specific interactions with anti-tetherin proteins, while maintaining virus restriction.
RESUMEN
The ß-amylase family in Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.
RESUMEN
Purpose: DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. Methods: We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO, PDE6B, PAX6, and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. Results: In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX-dependent transcription. Conclusions: Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.
Asunto(s)
Proteínas de Fase Aguda/química , Metilación de ADN , Epigénesis Genética , Proteínas de Homeodominio/química , Modelos Moleculares , Células Fotorreceptoras de Vertebrados/metabolismo , Transactivadores/química , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Secuencia de Bases , Cadáver , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Elementos de Nucleótido Esparcido Largo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Células Fotorreceptoras de Vertebrados/citología , Regiones Promotoras Genéticas , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transactivadores/genética , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
ß-Amylase3 (BAM3) is an enzyme that is essential for starch degradation in plant leaves and is also transcriptionally induced under cold stress. However, we recently reported that BAM3's enzymatic activity decreased in cold-stressed Arabidopsis leaves, although the activity of BAM1, a homologous leaf ß-amylase, was largely unaffected. This decrease in BAM3 activity may relate to the accumulation of starch reported in cold-stressed plants. The aim of this study was to explore the disparity between BAM3 transcript and activity levels under cold stress, and we present evidence suggesting BAM3 is being inhibited by post-translational modification. A mechanism of enzyme inhibition was suggested by observing that BAM3 protein levels remained unchanged under cold stress. Cold stress induces nitric oxide (NO) signaling, one result being alteration of protein activity by nitrosylation or glutathionylation through agents such as S-nitrosoglutathione (GSNO). To test whether NO induction correlates with inhibition of BAM3 in vivo, plants were treated with sodium nitroprusside, which releases NO, and a decline in BAM3 but not BAM1 activity was again observed. Treatment of recombinant BAM3 and BAM1 with GSNO caused significant, dose-dependent inhibition of BAM3 activity while BAM1 was largely unaffected. Site-directed mutagenesis, anti-glutathione Western blots, and mass spectrometry were then used to determine that in vitro BAM3 inhibition was caused by glutathionylation at cysteine 433. In addition, we generated a BAM1 mutant resembling BAM3 that was sensitive to GSNO inhibition. These findings demonstrate a differential response of two BAM paralogs to the Cys-modifying reagent GSNO and provide a possible molecular basis for reduced BAM3 activity in cold-stressed plants.
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Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Glutatión/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Frío , Cisteína/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Óxido Nítrico/fisiología , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , S-Nitrosoglutatión/farmacología , Transducción de Señal , Almidón/metabolismo , Estrés FisiológicoRESUMEN
The Arabidopsis (Arabidopsis thaliana) genome contains nine ß-amylase (BAM) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis BAM genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that BAM2 is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Potasio/metabolismo , beta-Amilasa/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Cinética , Modelos Moleculares , Hojas de la Planta/enzimología , Conformación Proteica , beta-Amilasa/química , beta-Amilasa/genéticaRESUMEN
A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.
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Miopatías Distales/genética , Filaminas/genética , Mutación del Sistema de Lectura , Factores de Intercambio de Guanina Nucleótido Rho/genética , Adulto , Biopsia , Miopatías Distales/diagnóstico por imagen , Miopatías Distales/patología , Femenino , Tamización de Portadores Genéticos , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Linaje , Proteínas Serina-Treonina Quinasas , Secuenciación del ExomaRESUMEN
BST-2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV-1 and other viruses by inhibiting viral spreading. Structurally, BST-2 is a homo-dimeric coiled-coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C-terminal membrane anchor of BST-2 is inserted into the budding virus while the N-terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST-2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST-2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated form to the cell-virus membrane bridging form. We observed that BST-2 did not transition as a rigid structure, but instead bent at positions with a reduced interface between the helices of the coiled-coil. The simulations for the human BST-2 were then compared with simulations on the mouse homolog, which has no apparent weak spots. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled-coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.
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Antígenos CD/química , Antígenos CD/metabolismo , Modelos Biológicos , Simulación de Dinámica Molecular , Animales , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Humanos , Ratones , Fosfatidiletanolaminas , Docilidad , Liberación del VirusRESUMEN
The intercalated disc of cardiac muscle embodies a highly-ordered, multifunctional network, essential for the synchronous contraction of the heart. Over 200 known proteins localize to the intercalated disc. The challenge now lies in their characterization as it relates to the coupling of neighboring cells and whole heart function. Using molecular, biochemical and imaging techniques, we characterized for the first time two small obscurin isoforms, obscurin-40 and obscurin-80, which are enriched at distinct locations of the intercalated disc. Both proteins bind specifically and directly to select phospholipids via their pleckstrin homology (PH) domain. Overexpression of either isoform or the PH-domain in cardiomyocytes results in decreased cell adhesion and size via reduced activation of the PI3K/AKT/mTOR pathway that is intimately linked to cardiac hypertrophy. In addition, obscurin-80 and obscurin-40 are significantly reduced in acute (myocardial infarction) and chronic (pressure overload) murine cardiac-stress models underscoring their key role in maintaining cardiac homeostasis. Our novel findings implicate small obscurins in the maintenance of cardiomyocyte size and coupling, and the development of heart failure by antagonizing the PI3K/AKT/mTOR pathway.
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Tamaño de la Célula , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Enfermedad Aguda , Empalme Alternativo/genética , Animales , Adhesión Celular , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Ratones Endogámicos C57BL , Proteínas Musculares/química , Proteínas Musculares/genética , Fosfatos de Fosfatidilinositol/metabolismo , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
Obscurins are cytoskeletal proteins with structural and regulatory roles encoded by OBSCN. Mutations in OBSCN are associated with the development of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Specifically, the R4344Q mutation present in immunoglobulin domain 58 (Ig58) was the first to be linked with the development of HCM. To assess the effects of R4344Q in vivo, we generated the respective knock-in mouse model. Mutant obscurins are expressed and incorporated normally into sarcomeres. The expression patterns of sarcomeric and Ca2+-cycling proteins are unaltered in sedentary 1-year-old knock-in myocardia, with the exception of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase 2 (SERCA2) and pentameric phospholamban whose levels are significantly increased and decreased, respectively. Isolated cardiomyocytes from 1-year-old knock-in hearts exhibit increased Ca2+-transients and Ca2+-load in the sarcoplasmic reticulum and faster contractility kinetics. Moreover, sedentary 1-year-old knock-in animals develop tachycardia accompanied by premature ventricular contractions, whereas 2-month-old knock-in animals subjected to pressure overload develop a DCM-like phenotype. Structural analysis revealed that the R4344Q mutation alters the distribution of electrostatic charges over the Ig58 surface, thus interfering with its binding capabilities. Consistent with this, wild-type Ig58 interacts with phospholamban modestly, and this interaction is markedly enhanced in the presence of R4344Q. Together, our studies demonstrate that under sedentary conditions, the R4344Q mutation results in Ca2+ deregulation and spontaneous arrhythmia, whereas in the presence of chronic, pathological stress, it leads to cardiac remodeling and dilation. We postulate that enhanced binding between mutant obscurins and phospholamban leads to SERCA2 disinhibition, which may underlie the observed pathological alterations.
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Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Señalización del Calcio , Calcio/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Cardiopatías/genética , Cardiopatías/metabolismo , Proteínas Musculares/genética , Mutación , Animales , Arritmias Cardíacas/diagnóstico , Modelos Animales de Enfermedad , Ecocardiografía , Electrocardiografía , Cardiopatías/diagnóstico , Espectroscopía de Resonancia Magnética , Ratones , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
The modification of proteins by ubiquitin-fold modifier 1 (UFM1) is implicated in many human diseases. Prior to conjugation, UFM1 undergoes activation by its cognate activating enzyme, UBA5. UBA5 is a non-canonical E1 activating enzyme that possesses an adenylation domain but lacks a distinct cysteine domain. Binding of UBA5 to UFM1 is mediated via an amino acid sequence, known as the UFM1-interacting sequence (UIS), located outside the adenylation domain that is required for UFM1 activation. However, the precise boundaries of the UIS are yet not clear and are still under debate. Here we revisit the interaction of UFM1 with UBA5 by determining the crystal structure of UFM1 fused to 13 amino acids of human UBA5. Using binding and activity assays, we found that His 336 of UBA5, previously not reported to be part of the UIS, occupies a negatively charged pocket on UFM1's surface. This His is involved in UFM1 binding and if mutated perturbs activation of UFM1. Surprisingly, we also found that the interaction between two UFM1 molecules mimics how the UIS binds UFM1. Specifically, UFM1 His 70 resembles UBA5 His336 and enters a negatively charged pocked on the other UFM1 molecule. Our results refine our understanding of UFM1-UBA5 binding.
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Proteínas/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Cinética , Unión Proteica , Proteínas/química , Enzimas Activadoras de Ubiquitina/químicaRESUMEN
Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s). Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation.
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Procesamiento Proteico-Postraduccional/fisiología , Proteínas/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinación/fisiología , Ubiquitinas/metabolismo , Humanos , Proteínas Ubiquitinadas/metabolismoRESUMEN
Single nucleotide polymorphisms (SNPs) in DNA can result in phenotypes where the biochemical basis may not be clear due to the lack of protein structures. With the growing number of modeling and simulation software available on the internet, students can now participate in determining how small changes in genetic information impact cellular protein structure and function. We have developed a modular series of activities to engage lab or lecture students in examining the basis for common phenotypes. The activities range from basic phenotype testing/observation to DNA sequencing and simulation of protein structure and dynamics. We provide as an example study of the bitterness receptor TAS2R38 and PTC tasting, however these activities are applicable to other SNPs or genomic variants with a direct connection to an observable phenotype. These activities are modular and can be mixed to meet the student capabilities and infrastructure availability. The complete sequence of activities will demonstrate the direct connection between gene structure, protein structure and organism function. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):526-536, 2016.
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Investigación Biomédica/educación , Biología Computacional/educación , Biología Molecular/educación , Polimorfismo de Nucleótido Simple/genética , Aprendizaje Basado en Problemas/métodos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Bases , Curriculum , Evaluación Educacional , Genómica/métodos , Humanos , Laboratorios , Modelos Biológicos , Modelos Moleculares , Simulación de Dinámica Molecular , Fenotipo , Conformación Proteica , Receptores Acoplados a Proteínas G/química , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de Ácido Nucleico , Programas Informáticos , Relación Estructura-Actividad , Estudiantes/psicología , Percepción del Gusto/fisiología , EnseñanzaRESUMEN
The pathogenic bacteria Legionella pneumophila is known to cause Legionnaires' Disease, a severe pneumonia that can be fatal to immunocompromised individuals and the elderly. Shohdy et al. identified the L. pneumophila vacuole sorting inhibitory protein VipF as a putative N-acetyltransferase based on sequence homology. We have characterized the basic structural and functional properties of VipF to confirm this original functional assignment. Sequence conservation analysis indicates two putative CoA-binding regions within VipF. Homology modeling and small angle X-ray scattering suggest a monomeric, dual-domain structure joined by a flexible linker. Each domain contains the characteristic beta-splay motif found in many acetyltransferases, suggesting that VipF may contain two active sites. Docking experiments suggest reasonable acetyl-CoA binding locations within each beta-splay motif. Broad substrate screening indicated that VipF is capable of acetylating chloramphenicol and both domains are catalytically active. Given that chloramphenicol is not known to be N-acetylated, this is a surprising finding suggesting that VipF is capable of O-acetyltransferase activity. Proteins 2016; 84:1422-1430. © 2016 Wiley Periodicals, Inc.