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1.
Mol Cell ; 57(2): 261-72, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25544560

RESUMO

Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.


Assuntos
Glicogênio/metabolismo , Doença de Lafora/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Oligossacarídeos/química , Fosfatos/química , Fosforilação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Tirosina Fosfatases não Receptoras/fisiologia
2.
Proc Natl Acad Sci U S A ; 111(20): 7272-7, 2014 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-24799671

RESUMO

Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 site-specific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.


Assuntos
Proteínas de Arabidopsis/química , Fosfatases de Especificidade Dupla/química , Glucanos/química , Glucose/química , Amido/química , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Carboidratos/química , Domínio Catalítico , Clonagem Molecular , Fosfatases de Especificidade Dupla/metabolismo , Fosfatos/química , Fosforilação , Folhas de Planta/metabolismo , Ligação Proteica , Conformação Proteica
3.
BMC Biochem ; 15: 8, 2014 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-24690255

RESUMO

BACKGROUND: The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. RESULTS: In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. CONCLUSIONS: Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.


Assuntos
Galinhas/imunologia , Escherichia coli/genética , Doença de Lafora/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Carboidratos/química , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/isolamento & purificação , Fosfatases de Especificidade Dupla/metabolismo , Glicogênio/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Masculino , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Ligação Proteica , Estabilidade Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência , Solubilidade
4.
PLoS One ; 8(7): e69523, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23922729

RESUMO

Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin's oligomerization.


Assuntos
Cisteína/metabolismo , Glucanos/metabolismo , Multimerização Proteica , Proteínas Tirosina Fosfatases não Receptoras/química , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Sequência de Aminoácidos , Animais , Metabolismo dos Carboidratos , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Mamíferos , Dados de Sequência Molecular , Mutagênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases
5.
Plant Cell ; 25(6): 2302-14, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23832589

RESUMO

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase like sex four2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fosfatases de Especificidade Dupla/metabolismo , Glucanos/metabolismo , Amido/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
6.
Planta ; 236(3): 867-77, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22729821

RESUMO

Terpenes comprise a distinct class of natural products that serve a diverse range of physiological functions, provide for interactions between plants and their environment and represent a resource for many kinds of practical applications. To better appreciate the importance of terpenes to overall growth and development, and to create a production capacity for specific terpenes of industrial interest, we have pioneered the development of strategies for diverting carbon flow from the native terpene biosynthetic pathways operating in the cytosol and plastid compartments of tobacco for the generation of specific classes of terpenes. In the current work, we demonstrate how difficult it is to divert the 5-carbon intermediates DMAPP and IPP from the mevalonate pathway operating in the cytoplasm for triterpene biosynthesis, yet diversion of the same intermediates from the methylerythritol phosphate pathway operating in the plastid compartment leads to the accumulation of very high levels of the triterpene squalene. This was assessed by the co-expression of an avian farnesyl diphosphate synthase and yeast squalene synthase genes targeting metabolism in the cytoplasm or chloroplast. We also evaluated the possibility of directing this metabolism to the secretory trichomes of tobacco by comparing the effects of trichome-specific gene promoters to strong, constitutive viral promoters. Surprisingly, when transgene expression was directed to trichomes, high-level squalene accumulation was observed, but overall plant growth and physiology were reduced up to 80 % of the non-transgenic controls. Our results support the notion that the biosynthesis of a desired terpene can be dramatically improved by directing that metabolism to a non-native cellular compartment, thus avoiding regulatory mechanisms that might attenuate carbon flux within an engineered pathway.


Assuntos
Ácido Mevalônico/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Triterpenos/metabolismo , Vias Biossintéticas , Citosol/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Engenharia Genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plastídeos/metabolismo , Regiões Promotoras Genéticas , Esqualeno/metabolismo
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