RESUMO
Archaea have diverse cell wall types, yet none are identical to bacterial peptidoglycan (murein). Methanogens Methanobacteria and Methanopyrus possess cell walls of pseudomurein, a structural analogue of murein. Pseudomurein differs from murein in containing the unique archaeal sugar N-acetyltalosaminuronic acid instead of N-acetylmuramic acid, ß-1,3 glycosidic bonds in place of ß-1,4 bonds and only l-amino acids in the peptide cross-links. We have determined crystal structures of methanogen pseudomurein peptide ligases (termed pMurE) from Methanothermus fervidus (Mfer762) and Methanothermobacter thermautotrophicus (Mth734) that are structurally most closely related to bacterial MurE peptide ligases. The homology of the archaeal pMurE and bacterial MurE enzymes is clear both in the overall structure and at the level of each of the three domains. In addition, we identified two UDP-binding sites in Mfer762 pMurE, one at the exterior surface of the interface of the N-terminal and middle domains, and a second site at an inner surface continuous with the highly conserved interface of the three domains. Residues involved in ATP binding in MurE are conserved in pMurE, suggesting that a similar ATP-binding pocket is present at the interface of the middle and the C-terminal domains of pMurE. The presence of pMurE ligases in members of the Methanobacteriales and Methanopyrales, that are structurally related to bacterial MurE ligases, supports the idea that the biosynthetic origins of archaeal pseudomurein and bacterial peptidoglycan cell walls are evolutionarily related.
Assuntos
Euryarchaeota , Peptidoglicano , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Archaea/metabolismo , Bactérias/metabolismo , Parede Celular/metabolismo , Euryarchaeota/metabolismo , Ligases/metabolismo , Peptídeo Sintases/metabolismo , Peptidoglicano/metabolismo , Açúcares/metabolismo , Difosfato de Uridina/análise , Difosfato de Uridina/metabolismoRESUMO
Flower sepals are critical for flower development and vary greatly in life span depending on their function post-pollination. Very little is known about what controls sepal longevity. Using a sepal senescence mutant screen, we identified two Arabidopsis mutants with delayed senescence directly connecting strigolactones with senescence regulation in a novel floral context that hitherto has not been explored. The mutations were in the strigolactone biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and in the strigolactone receptor gene DWARF14 (AtD14). The mutation in AtD14 changed the catalytic Ser97 to Phe in the enzyme active site, which is the first mutation of its kind in planta. The lesion in MAX1 was in the haem-iron ligand signature of the cytochrome P450 protein, converting the highly conserved Gly469 to Arg, which was shown in a transient expression assay to substantially inhibit the activity of MAX1. The two mutations highlighted the importance of strigolactone activity for driving to completion senescence initiated both developmentally and in response to carbon-limiting stress, as has been found for the more well-known senescence-associated regulators ethylene and abscisic acid. Analysis of transcript abundance in excised inflorescences during an extended night suggested an intricate relationship among sugar starvation, senescence, and strigolactone biosynthesis and signalling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Compostos Heterocíclicos com 3 Anéis , Lactonas , Reguladores de Crescimento de PlantasRESUMO
Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes-MAP3K7, encoding transforming growth factor ß (TGF-ß)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T [ p.Pro485Leu]) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C [p.Glu70Gln], c.299T>A [p.Val100Glu], and c.502G>C [p.Gly168Arg]). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex.
Assuntos
Testa/anormalidades , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Mutação/genética , Osteocondrodisplasias/genética , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Feminino , Filaminas/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , NF-kappa B/metabolismo , Osteocondrodisplasias/metabolismo , Fosforilação , Ligação Proteica , Multimerização ProteicaRESUMO
The crystal structure of UDP-N-acetylglucosamine 4-epimerase (UDP-GlcNAc 4-epimerase; WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N-terminal Rossmann-fold for nucleotide binding and an active site positioned in the C-terminus. UDP-GlcNAc 4-epimerase is a member of the short-chain dehydrogenases/reductases superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4-epimerases. The protein was co-crystallized with coenzyme NADH and UDP-N-acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non-native UDP sugar amongst archaea and was most likely incorporated from the E. coli expression host during purification of the recombinant enzyme.
Assuntos
Proteínas Arqueais/química , Carboidratos Epimerases/química , Methanobrevibacter/enzimologia , Modelos Moleculares , Uridina Difosfato Ácido N-Acetilmurâmico/química , Proteínas Arqueais/genética , Carboidratos Epimerases/genética , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/metabolismo , NAD/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.
Assuntos
Glucoquinase/química , Glucose/química , Dobramento de Proteína , Motivos de Aminoácidos , Animais , Glucoquinase/genética , Humanos , Camundongos , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn(2+) cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn(2+) cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH.
Assuntos
Evolução Molecular , Glicerolfosfato Desidrogenase/química , Glicerolfosfato Desidrogenase/genética , Lipídeos/biossíntese , Methanocaldococcus/enzimologia , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Cinética , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de Proteína , Zinco/metabolismoRESUMO
Recently, a newly identified autosomal recessive skeletal dysplasia was described characterized by calvarial abnormalities (including cranium bifidum, coronal, and lambdoid synostosis), oligodactyly, femoral bowing, narrow thorax, small pelvic bones, and radiohumeral synostosis. In the two families described, a more severe phenotype led to in utero lethality in three siblings while in a single patient in a second family the phenotype was sufficiently mild to allow survival to 5 months of age. The disorder is caused by biallelic missense mutations in CYP26B1, which encodes for a cytochrome P450 enzyme responsible for the catabolism of retinoic acid in a temporally and spatially restricted fashion during embryonic development. Here, we provide the third family affected by the disorder and the first affected individual to survive beyond infancy. This woman homozygous for c.1303G>A; p.(Gly435Ser) in CYP26B1, which was associated with multisutural synostosis, radiohumeral synostosis, normal bone mineral density, and apparent intellectual disability, a phenotype with significant similarities to Antley-Bixler and Pfeiffer syndromes. © 2016 Wiley Periodicals, Inc.
Assuntos
Acrocefalossindactilia/diagnóstico , Acrocefalossindactilia/genética , Alelos , Fenótipo de Síndrome de Antley-Bixler/diagnóstico , Fenótipo de Síndrome de Antley-Bixler/genética , Mutação , Ácido Retinoico 4 Hidroxilase/genética , Substituição de Aminoácidos , Análise Mutacional de DNA , Diagnóstico Diferencial , Fácies , Feminino , Homozigoto , Humanos , Modelos Moleculares , Fenótipo , Conformação Proteica , Ácido Retinoico 4 Hidroxilase/química , Crânio/anormalidades , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
We develop a video tracking method that utilizes an interpolation-based normalized cross-correlation approach to track the position of microscopic spherical particles in three dimensions. Subnanometer resolution is demonstrated. The method does not assume that the particle's image is radially symmetric, making it useful for determining the position when particles are close and their images overlap. This is demonstrated in a study of the electrostatic and hydrodynamic interactions between a pair of beads in dual laser tweezers traps.
RESUMO
Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.
Assuntos
Suturas Cranianas , Craniossinostoses , Sistema Enzimático do Citocromo P-450 , Tretinoína , Proteínas de Peixe-Zebra/genética , Animais , Diferenciação Celular , Suturas Cranianas/efeitos dos fármacos , Suturas Cranianas/embriologia , Suturas Cranianas/crescimento & desenvolvimento , Suturas Cranianas/patologia , Craniossinostoses/enzimologia , Craniossinostoses/genética , Craniossinostoses/patologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Modelos Animais de Doenças , Feminino , Morte Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Crescimento e Desenvolvimento/genética , Humanos , Camundongos , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Polimorfismo Genético/genética , Gravidez , Ácido Retinoico 4 Hidroxilase , Homologia de Sequência de Aminoácidos , Tretinoína/metabolismo , Tretinoína/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Methyl-coenzyme M reductase (MCR) is a multi-subunit (α2ß2γ2) enzyme responsible for methane formation via its unique F430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high-resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin-like domain assembled into an α + ß barrel-like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.
Assuntos
Oxirredutases , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/genética , Cristalografia por Raios X , Modelos Moleculares , Metano/metabolismo , Metano/química , Humanos , Conformação Proteica , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genéticaRESUMO
Methenyltetrahydromethanopterin cyclohydrolase (Mch) is involved in the methanogenesis pathway of archaea as a C1 unit carrier where N(5) -formyl-tetrahydromethanopterin is converted to methenyl-tetrahydromethanopterin. Mch from Methanobrevibacter ruminantium was cloned, purified, crystallized and its crystal structure solved at 1.37 Å resolution. A biologically active trimer, the enzyme is composed of two domains including an N-terminal domain of six α-helices encompassing a series of four ß-sheets and a predominantly anti-parallel ß-sheet at the C-terminus flanked on one side by α-helices. Sequence and structural alignments have helped identify residues involved in substrate binding and trimer formation.
Assuntos
Aminoidrolases/química , Methanobrevibacter/enzimologia , Proteínas Arqueais/química , Cristalografia por Raios XRESUMO
Remodeling of the cytoskeleton is central to the modulation of cell shape and migration. Filamin A, encoded by the gene FLNA, is a widely expressed protein that regulates re-organization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes and second messengers. We identified localized mutations in FLNA that conserve the reading frame and lead to a broad range of congenital malformations, affecting craniofacial structures, skeleton, brain, viscera and urogenital tract, in four X-linked human disorders: otopalatodigital syndrome types 1 (OPD1; OMIM 311300) and 2 (OPD2; OMIM 304120), frontometaphyseal dysplasia (FMD; OMIM 305620) and Melnick-Needles syndrome (MNS; OMIM 309350). Several mutations are recurrent, and all are clustered into four regions of the gene: the actin-binding domain and rod domain repeats 3, 10 and 14/15. Our findings contrast with previous observations that loss of function of FLNA is embryonic lethal in males but manifests in females as a localized neuronal migration disorder, called periventricular nodular heterotopia (PVNH; refs. 3-6). The patterns of mutation, X-chromosome inactivation and phenotypic manifestations in the newly described mutations indicate that they have gain-of-function effects, implicating filamin A in signaling pathways that mediate organogenesis in multiple systems during embryonic development.
Assuntos
Anormalidades Múltiplas/genética , Cromossomos Humanos X , Proteínas Contráteis/genética , Citoesqueleto/metabolismo , Ligação Genética , Proteínas dos Microfilamentos/genética , Mutação , Polimorfismo Genético , Alelos , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Análise Mutacional de DNA , Feminino , Filaminas , Humanos , Íntrons , Masculino , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Síndrome , Distribuição TecidualRESUMO
A novel murine enzyme, ADP-dependent glucokinase (ADPGK), has been shown to catalyse glucose phosphorylation using ADP as phosphoryl donor. The ancestral ADPGK gene appears to have been laterally transferred from Archaea early in metazoan evolution, but its biological role has not been established. Here, we undertake an initial investigation of the functional properties of human ADPGK in human tumour cell lines and specifically test the hypothesis that ADPGK might prime glycolysis using ADP under stress conditions such as hypoxia. Recombinant human ADPGK was confirmed to catalyse ADP-dependent glucose phosphorylation in vitro, with an apparent K (M) for glucose of 0.29 mM. Expression databases and western blotting of surgical samples demonstrated high expression in many human tissues, including tumours. Unlike hexokinase-2 (HK2), RNAi studies with exon arrays showed that ADPGK is not a transcriptional target of hypoxia inducible factor-1. Consistent with this, ADPGK protein was not upregulated by hypoxia or anoxia. Surprisingly, stable fivefold overexpression of ADPGK in H460 or HCT116 cells had no apparent effect on proliferation or glycolysis, and did not rescue clonogenicity or glycolysis when HK2 was suppressed by siRNA. Furthermore, suppression of ADPGK by siRNA did not cause detectable inhibition of glycolysis or cell killing by anoxia, although it did induce a statistically significant decrease in plating efficiency of H460 cells under aerobic conditions. Thus, human ADPGK catalyses ADP-dependent phosphorylation of glucose in vitro, but despite its high expression in human tumour cell lines it appears not to make a quantifiable contribution to glycolysis under the conditions evaluated.
Assuntos
Glucoquinase/genética , Glucoquinase/metabolismo , Glucose/metabolismo , Glicólise , Proteínas Recombinantes/metabolismo , Difosfato de Adenosina/metabolismo , Catálise , Hipóxia Celular/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Glucose/farmacologia , Células HCT116 , Células HT29 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Fosforilação , RNA Interferente Pequeno , Proteínas Recombinantes/genéticaRESUMO
Filamin A (FLNA) crosslinks F-actin and binds proteins consistent with roles integrating cell signalling and the cytoskeleton. FLNA missense mutations are associated with the otopalatodigital syndrome (OPD) spectrum of skeletal disorders, clustering in discrete domains. One cluster is found in the second calponin homology domain of the FLNA actin-binding domain (ABD), implicating this region as essential for mediating correct function. Here we show that OPD (FLNA E254K) fibroblast lysates have equivalent concentrations of FLNA compared with controls and that recombinant FLNA E254K ABD has increased in vitro F-actin binding (K(d) 13 microm) compared with wild type (WT; K(d) 48 microm). These observations are consistent with a gain-of-function mechanism for OPD. We have determined the crystal structures of the WT and E254K FLNA ABDs at 2.3 A resolution, revealing that they adopt similar closed conformations. The E254K mutation removes a conserved salt bridge but does not disrupt the ABD structure. The solution structures are also equivalent as determined by circular dichroism spectroscopy, but differential scanning fluorimetry denaturation showed reduced stability (decreased T(m) of 5.6 degrees C) for E254K relative to WT. Ex vivo characterization of E254K OPD patient fibroblasts revealed they have similar motility and adhesion as control cells, implying that many core functions mediated by FLNA are unaffected, consistent with OPD only affecting specific tissues despite FLNA being widely expressed. These data provide the first biochemical evidence for a gain-of-function mechanism for the OPD disorders, and mechanistically distinguishes them from the loss-of-function phenotypes that manifest as disorders of neuronal migration.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Movimento Celular/genética , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Sequência de Aminoácidos , Adesão Celular/genética , Proteínas Contráteis/química , Cristalografia por Raios X , Ossículos da Orelha/anormalidades , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Filaminas , Dedos/anormalidades , Humanos , Masculino , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Palato/anormalidades , Estrutura Terciária de Proteína , Síndrome , Dedos do Pé/anormalidadesRESUMO
Bacteria near-universally contain a cell wall sacculus of murein (peptidoglycan), the synthesis of which has been intensively studied for over 50 years. In striking contrast, archaeal species possess a variety of other cell wall types, none of them closely resembling murein. Interestingly though, one type of archaeal cell wall termed pseudomurein found in the methanogen orders Methanobacteriales and Methanopyrales is a structural analogue of murein in that it contains a glycan backbone that is cross-linked by a L-amino acid peptide. Here, we present taxonomic distribution, gene cluster and phylogenetic analyses that confirm orthologues of 13 bacterial murein biosynthesis enzymes in pseudomurein-containing methanogens, most of which are distantly related to their bacterial counterparts. We also present the first structure of an archaeal pseudomurein peptide ligase from Methanothermus fervidus DSM1088 (Mfer336) to a resolution of 2.5 Å and show that it possesses a similar overall tertiary three domain structure to bacterial MurC and MurD type murein peptide ligases. Taken together the data strongly indicate that murein and pseudomurein biosynthetic pathways share a common evolutionary history.
RESUMO
Bioengineered polyhydroxyalkanoate (PHA) spheres assembled in engineered bacteria are showing promising potential in protein immobilization for high-value applications. Here, we have designed innovative streamlined approaches to add functional proteins from complex mixtures (e.g., without prior purification) to bioengineered PHA spheres directly harnessing the specificity of the SpyTag/SpyCatcher mediated protein ligation. Escherichia coli was engineered to assemble PHA spheres displaying the SpyCatcher domain while simultaneously producing a SpyTagged target protein, which was in vivo specifically ligated to the PHA spheres. To further demonstrate the specificity of this ligation reaction, we incubated isolated SpyCatcher-coated PHA spheres with cell lysates containing SpyTagged target protein, which also resulted in specific ligation mediating surface functionalization. An even cruder approach was used by lysing a mixture of cells, either producing PHA spheres or target protein, which resulted in specific surface functionalization suggesting that ligation between the SpyCatcher-coated PHA spheres and the SpyTagged target proteins is highly specific. To expand the design space of this general modular approach toward programmable multifunctionalization, e.g., one-pot construction of immobilized multienzyme cascade systems on PHA spheres, we designed various recombinant bimodular PHA spheres utilizing alternative Tag/Catcher pairs (e.g., SnoopTag/SnoopCatcher and SdyTag/SdyCatcher systems). One of our bimodular PHA spheres resulted in simultaneous multifunctionalization of plain PHA spheres in one-step with two differently tagged proteins under in vitro and ex vivo reaction conditions while remaining functional. Our bimodular PHA spheres also showed high orthogonality with the non-target peptide tag and exhibited decent robustness against repeated freeze-thaw treatment. We demonstrated the utility of these approaches by using a fluorescent protein, a monomeric amylase, and a dimeric organophosphate hydrolase as target proteins. We established a versatile toolbox for dynamic functionalization of PHA spheres for biomedical and industrial applications.
RESUMO
Emerin is a ubiquitously expressed inner nuclear membrane protein of unknown function. Mutations in its gene give rise to X-linked Emery-Dreifuss muscular dystrophy (X-EDMD), a neuromuscular condition with an associated life-threatening cardiomyopathy. We have previously reported that emerin is phosphorylated in a cell cycle-dependent manner in human lymphoblastoid cell lines [Ellis et al. (1998) Aberrant intracellular targeting and cell cycle-dependent phosphorylation of emerin contribute to the EDMD phenotype. J. Cell Sci. 111, 781-792]. Recently, five residues in human emerin were identified as undergoing cell cycle-dependent phosphorylation using a Xenopus egg mitotic cytosol model system (Hirano et al. (2005) Dissociation of emerin from BAF is regulated through mitotic phosphorylation of emerin in a Xenopus egg cell-free system. J. Biol. Chem.280, 39 925-39 933). In the present paper, recombinant human emerin was purified from a baculovirus-Sf9 heterogeneous expression system, analyzed by protein mass spectrometry and shown to exist in at least four different phosphorylated species, each of which could be dephosphorylated by treatment with alkaline phosphatase. Further analysis identified three phosphopeptides with m/z values of 2191.9 and 2271.7 corresponding to the singly and doubly phosphorylated peptide 158-DSAYQSITHYRPVSASRSS-176, and a m/z of 2396.9 corresponding to the phosphopeptide 47-RLSPPSSSAASSYSFSDLNSTR-68. Sequence analysis confirmed that residue S49 was phosphorylated and also demonstrated that this residue was phosphorylated in interphase. Using an in vitro protein kinase A assay, we observed two phospho-emerin species, one of which was phosphorylated at residue S49. Protein kinase A is thus the first kinase that has been identified to specifically phosphorylate emerin. These results improve our understanding of the molecular mechanisms underlying X-EDMD and point towards possible signalling pathways involved in regulating emerin's functions.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Membrana/química , Proteínas Nucleares/química , Proteínas de Ancoragem à Quinase A , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Serina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Utrophin and dystrophin link cytoskeletal F-actin filaments to the plasmalemma. Genetic strategies to replace defective dystrophin with utrophin in individuals with muscular dystrophy requires full characterization of these proteins. Both contain homologous N-terminal actin-binding motifs composed of a pair of calponin-homology (CH) domains (CH1 and CH2) that are connected by spectrin-repeat modules to C-terminal membrane-binding sequences. Here, electron microscopy and 3D reconstruction of F-actin decorated with utrophin and dystrophin actin-binding constructs were performed using Utr261 (utrophin's CH domain pair), Utr416 (utrophin's CH domains and first spectrin-repeat) and Dys246 (dystrophin's CH domain pair). The lozenge-like utrophin CH domain densities localized to the upper surface of actin subdomain 1 and extended azimuthally over subdomain 2 toward subdomains 3 and 4. The cylinder-shaped spectrin-repeat was located at the end of the CH domain pair and was aligned longitudinally along the cleft between inner and outer actin domains, where tropomyosin is present when on thin filaments. The connection between the spectrin-repeat module and the CH domains defined the orientation of CH1 and CH2 on actin. Resolution of utrophin's CH domains and spectrin-repeats permitted docking of crystal structures into respective EM densities, leading to an atomic model where both CH and spectrin-domains bind actin. The CH domain-actin interaction for dystrophin was found to be more complex than for utrophin. Binding assays showed that Utr261 and Utr416 interacted with F-actin as monomers, whereas Dys246 appeared to associate as a dimer, consistent with a bilobed Dys246 structure observed on F-actin in electron microscope reconstructions. One of the lobes was similar in shape, position and orientation to the monomeric CH domains of Utr261, while the other lobe apparently represented a second set of CH domains in the dimeric Dys246. The extensive contact made by dystrophin on actin may be used in vivo to help muscles dissipate mechanical stress from the contractile apparatus to the extracellular matrix.
Assuntos
Actinas/metabolismo , Distrofina/metabolismo , Modelos Moleculares , Espectrina/metabolismo , Actinas/ultraestrutura , Animais , Sítios de Ligação , Distrofina/ultraestrutura , Escherichia coli/metabolismo , Microscopia Eletrônica , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrina/ultraestruturaRESUMO
UNLABELLED: Filamin A, the filamentous protein encoded by the X-linked gene FLNA, cross-links cytoskeletal actin into three-dimensional networks, facilitating its role as a signalling scaffold and a mechanosensor of extrinsic shear forces. Central to these functions is the ability of FLNA to form V-shaped homodimers through its C-terminal located filamin repeat 24. Additionally, many proteins that interact with FLNA have a binding site that includes the C-terminus of the protein. Here, a cohort of patients with mutations affecting this region of the protein is studied, with particular emphasis on the phenotype of male hemizygotes. Seven unrelated families are reported, with five exhibiting a typical female presentation of periventricular heterotopia (PH), a neuronal migration disorder typically caused by loss-of-function mutations in FLNA. One male presents with widespread PH consistent with previous male phenotypes attributable to hypomorphic mutations in FLNA. In stark contrast, two brothers are described with a mild PH presentation, due to a missense mutation (p.Gly2593Glu) inserting a large negatively charged amino acid into the hydrophobic dimerisation interface of FLNA. Co-immunoprecipitation, in vitro cross-linking studies and gel filtration chromatography all demonstrated that homodimerisation of isolated FLNA repeat 24 is abolished by this p.Gly2593Glu substitution but that extended FLNA(Gly2593Glu) repeat 16-24 constructs exhibit dimerisation. These observations imply that other interactions apart from those mediated by the canonical repeat 24 dimerisation interface contribute to FLNA homodimerisation and that mutations affecting this region of the protein can have broad phenotypic effects. KEY MESSAGES: ⢠Mutations in the X-linked gene FLNA cause a spectrum of syndromes. ⢠Genotype-phenotype correlations are emerging but still remain unclear. ⢠C-term mutations can confer male lethality, survival or connective tissue defects. ⢠Mutations leading to the latter affect filamin dimerisation. ⢠This deficit is compensated for by remotely acting domains elsewhere in FLNA.
Assuntos
Filaminas/genética , Heterotopia Nodular Periventricular/genética , Multimerização Proteica/genética , Sequência de Aminoácidos , Movimento Celular/genética , Feminino , Fibroblastos , Estudos de Associação Genética , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Fenótipo , Estrutura Terciária de ProteínaRESUMO
Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm--the size of three ribosomes and with a lumen capacity of 50 million Å(3). We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault's phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally.