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1.
Proc Natl Acad Sci U S A ; 114(34): E7197-E7204, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28784794

RESUMO

Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta, indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
2.
Plant Reprod ; 28(3-4): 143-51, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26111864

RESUMO

KEY MESSAGE: Establishment of apex-polarity. Elongation of the pollen tube is a highly coordinated process involving polarized secretion of cell wall and membrane materials to the apical region. We investigated changes in the localization of soluble NSF attachment proteins (SNAREs) in developing pollen grains and the pollen tube for transgenic Arabidopsis expressing pollen-specific plasma-membrane Qa-SNAREs (SYP124, 125 and 131) fused with the green fluorescent protein (GFP). The expression of SYP124 and SYP125 was firstly detected in the microspore nuclear membrane during pollen mitosis II. Although SYP124, 125 and 131 accumulated throughout the cytosol in the mature pollen grain, GFP-SYP124 and GFP-SYP125 were highly concentrated in the apical or subapical regions of the elongating pollen tube with slightly different localization patterns, whereas GFP-SYP131 was uniformly localized to the plasma membrane of the pollen tube. The apex-focused polarity of GFP-SYP125 was established coincident with formation of a Ca(2+) gradient before pollen germination. These results suggest that SNAREs function differentially in the same cells and that at least two distinct membrane transport pathways are involved in the pollen development and the pollen tube germination and elongation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Germinação , Membrana Nuclear/metabolismo , Pólen/crescimento & desenvolvimento , Proteínas Qa-SNARE/metabolismo , Arabidopsis/genética , Plantas Geneticamente Modificadas , Pólen/metabolismo , Tubo Polínico/crescimento & desenvolvimento
3.
Plant Signal Behav ; 10(3): e990847, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25897470

RESUMO

VAMP-associated proteins (VAPs) are highly conserved among eukaryotes. Here, we report a functional analysis of one of the VAPs, PVA31, and demonstrate its novel function on leaf senescence in Arabidopsis. The expression of PVA31 is highly induced in senescence leaves, and localizes to the plasma membrane as well as the ARA7-positive endosomes. Yeast two-hybrid analysis demonstrates that PVA31 is interacted with the plasma membrane localized-VAMP proteins, VAMP721/722/724 but not with the endosome-localized VAMPs, VAMP711 and VAMP727, indicating that PVA31 is associated with VAMP721/722/724 on the plasma membrane. Strong constitutive expression of PVA31 under the control of the Cauliflower mosaic virus 35S promoter induces the typical symptom of leaf senescence earlier than WT in normal growth and an artificially induced senescence conditions. In addition, the marker genes for the SA-mediated signaling pathways, PR-1, is promptly expressed with elicitor application. These data indicate that PVA31-overexpressing plants exhibit the early senescence phenotype in their leaves, and suggest that PVA31 is involved in the SA-mediated programmed cell death process during leaf senescence and PR-protein secretion during pathogen infection in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Resistência à Doença , Proteínas de Membrana/metabolismo , Doenças das Plantas , Folhas de Planta/metabolismo , Proteínas de Arabidopsis/genética , Caulimovirus , Morte Celular , Membrana Celular/metabolismo , Senescência Celular , Expressão Gênica , Fenótipo , Doenças das Plantas/virologia , Ácido Salicílico/metabolismo , Transdução de Sinais
4.
Hum Mol Genet ; 24(11): 3050-7, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25678555

RESUMO

In mitochondria, carbamoyl-phosphate synthetase 1 activity produces carbamoyl phosphate for urea synthesis, and deficiency results in hyperammonemia. Cytoplasmic carbamoyl-phosphate synthetase 2, however, is part of a tri-functional enzyme encoded by CAD; no human disease has been attributed to this gene. The tri-functional enzyme contains carbamoyl-phosphate synthetase 2 (CPS2), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which comprise the first three of six reactions required for de novo pyrimidine biosynthesis. Here we characterize an individual who is compound heterozygous for mutations in different domains of CAD. One mutation, c.1843-1G>A, results in an in-frame deletion of exon 13. The other, c.6071G>A, causes a missense mutation (p.Arg2024Gln) in a highly conserved residue that is essential for carbamoyl-phosphate binding. Metabolic flux studies showed impaired aspartate incorporation into RNA and DNA through the de novo synthesis pathway. In addition, CTP, UTP and nearly all UDP-activated sugars that serve as donors for glycosylation were decreased. Uridine supplementation rescued these abnormalities, suggesting a potential therapy for this new glycosylation disorder.


Assuntos
Aspartato Carbamoiltransferase/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Di-Hidro-Orotase/genética , Doenças Metabólicas/diagnóstico , Processamento de Proteína Pós-Traducional , Pirimidinas/biossíntese , Animais , Sequência de Bases , Células CHO , Pré-Escolar , Cricetinae , Cricetulus , Análise Mutacional de DNA , Estudos de Associação Genética , Glicosilação , Heterozigoto , Humanos , Masculino , Doenças Metabólicas/genética , Mutação de Sentido Incorreto , Deleção de Sequência
5.
Plant Physiol ; 167(4): 1361-73, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25699591

RESUMO

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Autofagia , Citoplasma/metabolismo , Endossomos/metabolismo , Genes Reporter , Modelos Biológicos , Mutação , Fenótipo , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Ubiquitinadas/metabolismo , Proteínas de Transporte Vesicular/genética
6.
Biochem Biophys Res Commun ; 453(2): 220-8, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24931670

RESUMO

Mannose is a simple sugar with a complex life. It is a welcome therapy for genetic and acquired human diseases, but it kills honeybees and blinds baby mice. It could cause diabetic complications. Mannose chemistry, metabolism, and metabolomics in cells, tissues and mammals can help explain these multiple systemic effects. Mannose has good, bad or ugly outcomes depending on its steady state levels and metabolic flux. This review describes the role of mannose at cellular level and its impact on organisms.


Assuntos
Manose/metabolismo , Animais , Defeitos Congênitos da Glicosilação/dietoterapia , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Carboidratos da Dieta/administração & dosagem , Carboidratos da Dieta/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Manose/administração & dosagem , Manose/química , Manose-6-Fosfato Isomerase/deficiência , Manose-6-Fosfato Isomerase/genética , Manose-6-Fosfato Isomerase/metabolismo , Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Metabolômica , Camundongos , Fosfotransferases (Fosfomutases)/deficiência , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Gravidez , Peixe-Zebra
7.
J Allergy Clin Immunol ; 133(5): 1400-9, 1409.e1-5, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24589341

RESUMO

BACKGROUND: Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. OBJECTIVE: We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment. METHODS: Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations. RESULTS: Marked atopy and autoimmunity were associated with increased T(H)2 and T(H)17 cytokine production by CD4(+) T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8(+) T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate-N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation. CONCLUSIONS: Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination.


Assuntos
Doenças Autoimunes/genética , Transtornos Cognitivos/genética , Imunodeficiência de Variável Comum/genética , Doenças Genéticas Inatas/genética , Hipersensibilidade/genética , Mutação , Fosfoglucomutase/genética , Doenças Autoimunes/enzimologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Criança , Pré-Escolar , Transtornos Cognitivos/enzimologia , Transtornos Cognitivos/imunologia , Transtornos Cognitivos/patologia , Imunodeficiência de Variável Comum/enzimologia , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/patologia , Família , Feminino , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Humanos , Hipersensibilidade/enzimologia , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Masculino , Linhagem , Fosfoglucomutase/imunologia , Fosfoglucomutase/metabolismo , Células Th17/enzimologia , Células Th17/imunologia , Células Th17/patologia , Células Th2/enzimologia , Células Th2/imunologia , Células Th2/patologia , Adulto Jovem
8.
Plant Cell Physiol ; 55(4): 790-800, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24642714

RESUMO

Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Qa-SNARE/metabolismo , Actinas/metabolismo , Arabidopsis/efeitos dos fármacos , Brefeldina A/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Mutação/genética , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE/metabolismo , Tiazolidinas/farmacologia
9.
N Engl J Med ; 370(6): 533-42, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24499211

RESUMO

BACKGROUND: Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS: Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS: Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS: Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).


Assuntos
Glucofosfatos/genética , Doença de Depósito de Glicogênio/genética , Fenótipo , Fosfoglucomutase/genética , Galactose/uso terapêutico , Genes Recessivos , Glucose/metabolismo , Glucofosfatos/metabolismo , Doença de Depósito de Glicogênio/dietoterapia , Doença de Depósito de Glicogênio/metabolismo , Glicoproteínas/biossíntese , Glicosilação , Humanos , Masculino , Mutação , Fosfoglucomutase/metabolismo , RNA Mensageiro/análise
10.
FASEB J ; 28(4): 1854-69, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24421398

RESUMO

Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (~15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1-2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ~50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.


Assuntos
Cegueira/etiologia , Suplementos Nutricionais/toxicidade , Manose-6-Fosfato Isomerase/metabolismo , Manose/toxicidade , Animais , Cegueira/genética , Cegueira/metabolismo , Western Blotting , Células Cultivadas , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Olho/embriologia , Olho/crescimento & desenvolvimento , Olho/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Manose/sangue , Manose/metabolismo , Manose-6-Fosfato Isomerase/genética , Manosefosfatos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Placenta/efeitos dos fármacos , Placenta/embriologia , Placenta/metabolismo , Gravidez
11.
J Biol Chem ; 289(10): 6751-61, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24407290

RESUMO

Mannose in N-glycans is derived from glucose through phosphomannose isomerase (MPI, Fru-6-P ↔ Man-6-P) whose deficiency causes a congenital disorder of glycosylation (CDG)-Ib (MPI-CDG). Mannose supplements improve patients' symptoms because exogenous mannose can also directly contribute to N-glycan synthesis through Man-6-P. However, the quantitative contributions of these and other potential pathways to glycosylation are still unknown. We developed a sensitive GC-MS-based method using [1,2-(13)C]glucose and [4-(13)C]mannose to measure their contribution to N-glycans synthesized under physiological conditions (5 mm glucose and 50 µm mannose). Mannose directly provides ∼10-45% of the mannose found in N-glycans, showing up to a 100-fold preference for mannose over exogenous glucose based on their exogenous concentrations. Normal human fibroblasts normally derive 25-30% of their mannose directly from exogenous mannose, whereas MPI-deficient CDG fibroblasts with reduced glucose flux secure 80% of their mannose directly. Thus, both MPI activity and exogenous mannose concentration determine the metabolic flux into the N-glycosylation pathway. Using various stable isotopes, we found that gluconeogenesis, glycogen, and mannose salvaged from glycoprotein degradation do not contribute mannose to N-glycans in fibroblasts under physiological conditions. This quantitative assessment of mannose contribution and its metabolic fate provides information that can help bolster therapeutic strategies for treating glycosylation disorders with exogenous mannose.


Assuntos
Glicoproteínas/metabolismo , Manose/metabolismo , Células Cultivadas , Fibroblastos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Gluconeogênese , Glucose/metabolismo , Glicogênio/metabolismo , Glicosilação , Humanos , Proteólise
12.
J Biol Chem ; 286(45): 39431-8, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-21949237

RESUMO

Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos.


Assuntos
Defeitos Congênitos da Glicosilação/tratamento farmacológico , Defeitos Congênitos da Glicosilação/enzimologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/enzimologia , Manose-6-Fosfato Isomerase/antagonistas & inibidores , Fosfotransferases (Fosfomutases)/genética , Animais , Defeitos Congênitos da Glicosilação/genética , Inibidores Enzimáticos/uso terapêutico , Glicosilação/efeitos dos fármacos , Células HeLa , Humanos , Manose/genética , Manose/metabolismo , Manose-6-Fosfato Isomerase/genética , Manose-6-Fosfato Isomerase/metabolismo , Manosefosfatos/genética , Manosefosfatos/metabolismo , Mutação , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
13.
J Med Chem ; 54(10): 3661-8, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21539312

RESUMO

We report the discovery and validation of a series of benzoisothiazolones as potent inhibitors of phosphomannose isomerase (PMI), an enzyme that converts mannose-6-phosphate (Man-6-P) into fructose-6-phosphate (Fru-6-P) and, more importantly, competes with phosphomannomutase 2 (PMM2) for Man-6-P, diverting this substrate from critical protein glycosylation events. In congenital disorder of glycosylation type Ia, PMM2 activity is compromised; thus, PMI inhibition is a potential strategy for the development of therapeutics. High-throughput screening (HTS) and subsequent chemical optimization led to the identification of a novel class of benzoisothiazolones as potent PMI inhibitors having little or no PMM2 inhibition. Two complementary synthetic routes were developed, enabling the critical structural requirements for activity to be determined, and the compounds were subsequently profiled in biochemical and cellular assays to assess efficacy. The most promising compounds were also profiled for bioavailability parameters, including metabolic stability, plasma stability, and permeability. The pharmacokinetic profile of a representative of this series (compound 19; ML089) was also assessed, demonstrating the potential of this series for in vivo efficacy when dosed orally in disease models.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Manose-6-Fosfato Isomerase/antagonistas & inibidores , Tiazóis/química , Administração Oral , Química Farmacêutica/métodos , Técnicas de Química Combinatória/métodos , Defeitos Congênitos da Glicosilação/genética , Desenho de Drogas , Humanos , Concentração Inibidora 50 , Modelos Químicos , Permeabilidade , Relação Estrutura-Atividade
14.
Mol Cancer Res ; 9(2): 133-48, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21228116

RESUMO

The tumor microenvironment plays an important role in modulating tumor progression. Earlier, we showed that S100A8/A9 proteins secreted by myeloid-derived suppressor cells (MDSC) present within tumors and metastatic sites promote an autocrine pathway for accumulation of MDSC. In a mouse model of colitis-associated colon cancer, we also showed that S100A8/A9-positive cells accumulate in all regions of dysplasia and adenoma. Here we present evidence that S100A8/A9 interact with RAGE and carboxylated glycans on colon tumor cells and promote activation of MAPK and NF-κB signaling pathways. Comparison of gene expression profiles of S100A8/A9-activated colon tumor cells versus unactivated cells led us to identify a small cohort of genes upregulated in activated cells, including Cxcl1, Ccl5 and Ccl7, Slc39a10, Lcn2, Zc3h12a, Enpp2, and other genes, whose products promote leukocyte recruitment, angiogenesis, tumor migration, wound healing, and formation of premetastatic niches in distal metastatic organs. Consistent with this observation, in murine colon tumor models we found that chemokines were upregulated in tumors, and elevated in sera of tumor-bearing wild-type mice. Mice lacking S100A9 showed significantly reduced tumor incidence, growth and metastasis, reduced chemokine levels, and reduced infiltration of CD11b(+)Gr1(+) cells within tumors and premetastatic organs. Studies using bone marrow chimeric mice revealed that S100A8/A9 expression on myeloid cells is essential for development of colon tumors. Our results thus reveal a novel role for myeloid-derived S100A8/A9 in activating specific downstream genes associated with tumorigenesis and in promoting tumor growth and metastasis.


Assuntos
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Neoplasias do Colo/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Transdução de Sinais/genética , Animais , Células da Medula Óssea/patologia , Calgranulina A/genética , Calgranulina B/genética , Linhagem Celular Tumoral , Proliferação de Células , Quimiocinas/metabolismo , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica , Polissacarídeos/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Ligação Proteica , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Microambiente Tumoral/genética
15.
Plant Cell Physiol ; 50(2): 280-9, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19098073

RESUMO

Membrane trafficking to the plasma membrane (PM) is a highly organized process which enables plant cells to build up their bodies. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) genes, which encode the proteins involved in membrane trafficking, are much more abundant in the Arabidopsis genome than in that of any other eukaryote. We have previously shown that a large number of SNARE molecules in the Arabidopsis cell are localized predominantly on the PM. In the present study, in order to elucidate the physiological function of each PM-localized SNARE, we analyzed the spatiotemporal expression profiling of nine SYP1s that are resident in the PM of Arabidopsis, and used the information thus acquired to generate transgenic Arabidopsis plants expressing green fluorescent protein-fused Qa-SNAREs under control of their authentic promoters. Among the nine SYP1s, only SYP132 is expressed ubiquitously in all tissues throughout plant development. The expression patterns of the other SYP1s, in contrast, are tissue specific, and all different from one another. A particularly noteworthy example is SYP123, which is predominantly expressed in root hair cells during root development, and shows a focal accumulation pattern at the tip region of root hairs. These results suggest that SYP132 is involved in constitutive membrane trafficking to the PM throughout plant development, while the other SYP1s are involved in membrane trafficking events such as root formation or tip growth of root hair, with some redundancy.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Proteínas Qa-SNARE/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
16.
Glycobiology ; 15(11): 1084-93, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16079417

RESUMO

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.


Assuntos
Defeitos Congênitos da Glicosilação/metabolismo , Fibroblastos/metabolismo , Manosefosfatos/farmacologia , Fosfatos Açúcares/farmacologia , Configuração de Carboidratos , Metabolismo dos Carboidratos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Relação Dose-Resposta a Droga , Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Manosefosfatos/síntese química , Manosefosfatos/química , Mutação , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Fosfatos Açúcares/síntese química , Fosfatos Açúcares/química , Fatores de Tempo
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