Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
1.
EMBO J ; 40(20): e107766, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34516001

RESUMO

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.


Assuntos
Glicoesfingolipídeos/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Brefeldina A/farmacologia , Ceramidas/metabolismo , Toxina da Cólera/farmacologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Toxina Shiga/farmacologia
2.
Mol Cell ; 55(1): 123-37, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24910095

RESUMO

NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.


Assuntos
Replicação do DNA , Coativadores de Receptor Nuclear/fisiologia , Origem de Replicação , Animais , Células Cultivadas , Senescência Celular , Células HeLa , Humanos , Camundongos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevis
3.
EMBO J ; 36(12): 1736-1754, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28495678

RESUMO

Sphingolipids are membrane lipids globally required for eukaryotic life. The sphingolipid content varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Due to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. The basis for maintaining distinct subcellular sphingolipid levels in the presence of membrane trafficking and metabolic fluxes is only partially understood. Here, we describe a homeostatic regulatory circuit that controls sphingolipid levels at the trans-Golgi network (TGN). Specifically, we show that sphingomyelin production at the TGN triggers a signalling pathway leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol and sphingolipid transport to the trans-Golgi network, PtdIns(4)P consumption interrupts this transport in response to excessive sphingomyelin production. Based on this evidence, we envisage a model where this homeostatic circuit maintains a constant lipid composition in the trans-Golgi network and post-Golgi compartments, thus counteracting fluctuations in the sphingolipid biosynthetic flow.


Assuntos
Fosfatidilinositóis/metabolismo , Esfingolipídeos/metabolismo , Rede trans-Golgi/metabolismo , Células HeLa , Homeostase , Humanos , Modelos Biológicos
4.
Cell Mol Life Sci ; 75(17): 3283-3296, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29564477

RESUMO

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.


Assuntos
Antígenos de Neoplasias/química , Bioquímica/métodos , Biofísica/métodos , Anidrase Carbônica IX/química , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sítios de Ligação/genética , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Domínio Catalítico , Dicroísmo Circular , Progressão da Doença , Humanos , Espectroscopia de Ressonância Magnética , Neoplasias/enzimologia , Neoplasias/patologia , Conformação Proteica , Proteoglicanas/metabolismo , Relação Estrutura-Atividade
5.
Amino Acids ; 46(2): 279-88, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23604465

RESUMO

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (ß-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the ß-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.


Assuntos
Aminoácido Oxirredutases/química , Proteínas Fúngicas/química , Glucose/química , Produtos Finais de Glicação Avançada/química , Lactoglobulinas/química , Leite/química , Sequência de Aminoácidos , Animais , Armazenamento de Alimentos , Frutosamina/química , Concentração de Íons de Hidrogênio , Lactose/química , Dados de Sequência Molecular , Pasteurização
6.
J Enzyme Inhib Med Chem ; 29(4): 500-4, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23895630

RESUMO

C3 and C4 plant carbonic anhydrases (CAs) are zinc-enzymes that catalyze the reversible hydration of CO2. They are sub-divided in three classes: α, ß and γ, being distributed between both photosynthetic subtypes. The C4 dicotyledon species Flaveria bidentis (L.) "Kuntze" contains a small gene family encoding three distinct ß-CAs, named FbiCA1, FbiCA2 and FbiCA3. We have expressed and purified recombinant FbiCA1, which is localized in the chloroplast where it is thought to play a role in lipid biosynthesis and antioxidant activity, and biochemically characterized it by spectroscopic and inhibition experiments. FbiCA1 is a compact octameric protein that is moderately inhibited by carboxylate molecules. Surprisingly, pyruvate, but not lactate, did not inhibit FbiCA1 at concentrations up to 10 mM, suggesting that its capacity to tolerate high pyruvate concentration reflects the high concentration of pyruvate in the chloroplasts of bundle-sheath and mesophyll cells involved in C4 photosynthesis.


Assuntos
Antioxidantes/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Ácidos Carboxílicos/farmacologia , Flaveria/enzimologia , Sequência de Aminoácidos , Antioxidantes/isolamento & purificação , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/isolamento & purificação , Ácidos Carboxílicos/química , Relação Dose-Resposta a Droga , Lipídeos/biossíntese , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
7.
J Exp Clin Cancer Res ; 43(1): 137, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38711119

RESUMO

BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models.  CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.


Assuntos
Oxirredutases do Álcool , Proteínas de Ligação a DNA , Melanoma , Humanos , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/genética , Animais , Camundongos , Melanoma/tratamento farmacológico , Melanoma/patologia , Melanoma/metabolismo , Melanoma/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Bioorg Med Chem Lett ; 23(6): 1626-30, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23414801

RESUMO

Several ß-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C4 plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO2 hydrase catalytic activity (kcat of 1.2×10(5) and kcat/Km of 7.5×10(6)M(-1)×s(-1)) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (KIs in the range of 4-60µM). Such inhibitors may be used as tools to better understand the role of various ß-CA isoforms in photosynthesis.


Assuntos
Ânions/química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Flaveria/enzimologia , Sequência de Aminoácidos , Ânions/metabolismo , Dióxido de Carbono/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/classificação , Anidrases Carbônicas/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/classificação , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
Biochem Biophys Res Commun ; 420(3): 542-6, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22440394

RESUMO

Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes.


Assuntos
Psychrobacter/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Esterol Esterase/biossíntese , Esterol Esterase/química , Dicroísmo Circular , Estabilidade Enzimática , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Luz , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Espalhamento de Radiação , Solubilidade , Esterol Esterase/isolamento & purificação , Trealose/farmacologia
10.
Cancers (Basel) ; 14(21)2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36358629

RESUMO

Intracellular mono-ADP-ribosyltransferases (mono-ARTs) catalyze the covalent attachment of a single ADP-ribose molecule to protein substrates, thus regulating their functions. PARP10 is a soluble mono-ART involved in the modulation of intracellular signaling, metabolism and apoptosis. PARP10 also participates in the regulation of the G1- and S-phase of the cell cycle. However, the role of this enzyme in G2/M progression is not defined. In this study, we found that genetic ablation, protein depletion and pharmacological inhibition of PARP10 cause a delay in the G2/M transition of the cell cycle. Moreover, we found that the mitotic kinase Aurora-A, a previously identified PARP10 substrate, is actively mono-ADP-ribosylated (MARylated) during G2/M transition in a PARP10-dependent manner. Notably, we showed that PARP10-mediated MARylation of Aurora-A enhances the activity of the kinase in vitro. Consistent with an impairment in the endogenous activity of Aurora-A, cells lacking PARP10 show a decreased localization of the kinase on the centrosomes and mitotic spindle during G2/M progression. Taken together, our data provide the first evidence of a direct role played by PARP10 in the progression of G2 and mitosis, an event that is strictly correlated to the endogenous MARylation of Aurora-A, thus proposing a novel mechanism for the modulation of Aurora-A kinase activity.

11.
Bioorg Med Chem Lett ; 20(17): 5023-6, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20688517

RESUMO

Human carbonic anhydrase VII (hCA VII) is a cytosolic member of the alpha-CA family. This enzyme is mainly localized in a number of brain tissues such as the cortex, hippocampus and thalamus and has been noted for its contribution in generating neuronal excitation and seizures. Recently, it has been also proposed that hCA VII may be involved in the control of neuropathic pain, thus its inhibition may offer a new approach in designing pain killers useful for combating neuropathic pain. We report here the X-ray crystallographic structure of a mutated form of human CA VII in complex with acetazolamide, a classical sulfonamide inhibitor. These crystallographic studies provide important implications for the rational drug design of selective CA inhibitors with clinical applications.


Assuntos
Acetazolamida/química , Anidrases Carbônicas/química , Mutação , Sequência de Aminoácidos , Anidrases Carbônicas/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
12.
Protein Expr Purif ; 59(2): 302-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18420420

RESUMO

PLD's (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx(4)Dx(6)GSxN motifs. PLD1 interacts with the small phosphoprotein PED-PEA15 by an unknown mechanism that, by enhancing PLD1 stability, apparently increases its enzymatic activity; the minimum interacting region of PLD1 was previously identified as spanning residues 712-1074 (D4 region). Since the D4/PED-PEA15 interaction has been claimed to be one of the multiple molecular events that can trigger type 2 diabetes, we purified the two recombinant proteins to study in vitro this binding by both ELISA and SPR techniques. Whilst PED-PEA15 was easily expressed and purified, expression of recombinant D4 was more problematic and only the fusion protein with Thioredoxin A and a six Histidine Tag (Trx-His(6)-D4) demonstrated sufficient stability for further characterization. We have found that Trx-His(6)-D4 is present as two different oligomeric forms, though only the monomeric variant is able to interact with PED-PEA15. All these findings may have important implications for both the mechanisms of phospholipase activity and PED-PEA15 regulative functions.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Fosfolipase D/química , Fosfolipase D/isolamento & purificação , Fosfoproteínas/química , Proteínas Reguladoras de Apoptose , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Fosfolipase D/biossíntese , Estrutura Terciária de Proteína/genética , Ressonância de Plasmônio de Superfície
13.
Nucleic Acids Res ; 30(22): 4945-51, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12433998

RESUMO

The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2-His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15-78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2-His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Aminoácidos Básicos/fisiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Cisteína/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Histidina/metabolismo , Dados de Sequência Molecular , Deleção de Sequência , Fatores de Transcrição/genética , Dedos de Zinco
15.
Biochimie ; 97: 114-20, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24140957

RESUMO

L-Histidinol dehydrogenase from Brucella suis (BsHDH) is an enzyme involved in the histidine biosynthesis pathway which is absent in mammals, thus representing a very interesting target for the development of anti-Brucella agents. In this paper we report the crystallographic structure of a mutated form of BsHDH both in its unbound form and in complex with a nanomolar inhibitor. These studies provide the first structural background for the rational design of potent HDH inhibitors, thus offering new hints for clinical applications.


Assuntos
Oxirredutases do Álcool/química , Antibacterianos/química , Proteínas de Bactérias/química , Brucella suis/química , Butanonas/química , Inibidores Enzimáticos/química , Imidazóis/química , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Brucella suis/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Histidina/química , Histidina/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutação , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
Biochimie ; 94(5): 1232-41, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22381359

RESUMO

Carbonic anhydrases (CAs) catalyze with high efficiency the reversible hydration of carbon dioxide, an essential reaction for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone resorption. Diatoms, which are one of the most common types of phytoplankton and are widespread in oceans, possess CAs fundamental for acquisition of inorganic carbon. Recently, in the marine diatom Thalassiosira weissflogii a novel enzyme, CDCA1, naturally using Cd in its active site, has been isolated and categorized in a new CA class, namely zeta-CA. This enzyme, which consists of three repeats (R1, R2 and R3), is a cambialistic carbonic anhydrase that can spontaneously exchange Zn or Cd at its active centre, presumably an adaptative advantage for diatoms that grow fast in the metal-poor environment of the surface ocean. In this paper we completed the characterization of this enzyme, reporting the X-ray structure of the last repeat, CDCA1-R3 in its cadmium-bound form, and presenting a model of the full length protein obtained by docking approaches. Results show that CDCA1 has a quite compact not symmetric structure, characterized by two covalently linked R1-R2 and R2-R3 interfaces and a small non-covalent R1-R3 interface. The three dimensional arrangement shows that most of the non-conserved aminoacids of the three repeats are located at the interface regions and that the active sites are far from each other and completely accessible to the substrate. Finally, a detailed inhibition study of CDCA1-R3 repeat in both cadmium- and zinc- bound form has been performed with sulfonamides and sulfamates derivatives. The results have been compared with those previously reported for other CA classes, namely alpha- and beta-classes, and correlated with the structural features of these enzymes.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Diatomáceas/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
17.
Mol Biosyst ; 6(10): 2039-48, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20714510

RESUMO

The interaction of Phospholipase D1 (PLD1) by its C-terminal domain D4 with PED/PEA15 has been indicated as a target for type 2 diabetes. PED/PEA15 is overexpressed in several tissues of individuals affected by type 2 diabetes and its overexpression in intact cells and in transgenic animal models impairs insulin regulation of glucose transport by a mechanism mediated by the interaction with D4 and the consequent increase of protein kinase C-alpha activity. Expression of D4 or administration of a peptide mimicking the PED/PEA15 region involved in this interaction to cells stably overexpressing PED/PEA15 reduces its interaction with PLD1, thereby lowering PKC-alpha activation and restoring normal glucose transport mediated by PKC-zeta. By using D4 deletion mutants, we have restricted the PLD1 region involved in PED/PEA15 interaction to an N-terminal fragment named D4alpha (residues 712-818). This region binds PED/PEA15 with the same efficacy as D4 (K(D) approximately 0.7 microM) and, when transfected in different PED/PEA15-overexpressing cells, it is able to reduce PKC-alpha activity and to restore the sensitivity of PKC-zeta to insulin stimulation, independently of the PI3K/Akt signalling. We also show that the effective disruption of the PED/PEA15-PLD1 interaction can restore the normal ERK1/2 signalling. Finally, using a set of overlapping peptides that cover the D4alpha region, we have further restricted the shortest PED/PEA15-binding site to a segment encompassing residues 762-801, suggesting that a quite limited binding interface mostly contributes to the interaction and can thus be a selective target for the design of effective antagonists.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfolipase D/metabolismo , Fosfoproteínas/metabolismo , Proteínas Reguladoras de Apoptose , Sequência de Bases , Primers do DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Mutagênese Sítio-Dirigida , Fosfolipase D/química , Fosfoproteínas/química , Ligação Proteica , Transdução de Sinais
18.
Endocrinology ; 151(4): 1948-58, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20160132

RESUMO

We report here the mapping of a chromosomal region responsible for strain-specific development of congenital hypothyroidism in mice heterozygous for null mutations in genes encoding Nkx2-1/Titf1 and Pax8. The two strains showing a differential predisposition to congenital hypothyroidism contain several single-nucleotide polymorphisms in this locus, one of which leads to a nonsynonymous amino acid change in a highly conserved region of Dnajc17, a member of the type III heat-shock protein-40 (Hsp40) family. We demonstrate that Dnajc17 is highly expressed in the thyroid bud and had an essential function in development, suggesting an important role of this protein in organogenesis and/or function of the thyroid gland.


Assuntos
Hipotireoidismo Congênito/genética , Predisposição Genética para Doença/genética , Proteínas de Choque Térmico HSP40/genética , Glândula Tireoide/anormalidades , Animais , Western Blotting , Células Cultivadas , Mapeamento Cromossômico , Cromossomos de Mamíferos/genética , Hipotireoidismo Congênito/metabolismo , Estudos de Associação Genética , Proteínas de Choque Térmico HSP40/metabolismo , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/metabolismo , Tireotropina/sangue
19.
Mol Immunol ; 46(16): 3300-9, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19699527

RESUMO

FcvarepsilonRIalpha found on the surface of mast cells and basophiles mediates allergic diseases, anaphylaxis and asthma through binding of IgE. Disrupting this interaction with anti-IgE mAbs has proven an efficient approach to control these diseases. The crystallographic structure of the complex formed between the IgE-Fc and FcvarepsilonRIalpha extracellular domain has shown that recognition is mediated by residues in the second Ig-like domain of the receptor (D2) and in the loop connecting the D1 and D2 domains. In an attempt to obtain specific IgE antagonists, we have designed and prepared a polypeptide named IgE-Trap that partially reproduces the IgE receptor-binding sites and binds with micromolar affinity to soluble IgE. The polypeptide contains loops C'-E [residues 129-134] and F-G [residues 151-161] from the D2 domain joined by a linker, and loop B-C [residues 110-113]. Peptide binding to IgE has been assessed by SPR analyses and the data fit with a biphasic model of interaction, in agreement with the two-site mechanism reported for the native receptor. The polypeptide binds to immobilized IgE in a dose-dependent manner with a K(D) estimated to be around 6muM, while it does not recognize IgG nor IgA. Polypeptide sub-domains involved in IgE binding have also been defined, showing that loop C'-E connected to loop B-C, but also the isolated loop B-C alone suffice to bind immunoglobulins E with high selectively though with reduced affinity compared to IgE-Trap. ELISA and cytometric assays on RBL2H3 cells demonstrate that the interacting peptides are able to displace the binding of IgE to receptor, confirming affinity and specificity of these ligands and suggesting a potential application as modulators of disorders associated with inappropriate IgE production.


Assuntos
Imunoglobulina E/química , Peptídeos/química , Receptores de IgE/química , Sítios de Ligação , Linhagem Celular , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/metabolismo , Imunoglobulina E/metabolismo , Ligantes , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de IgE/genética , Receptores de IgE/metabolismo
20.
Chem Biol Drug Des ; 73(5): 483-93, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19366357

RESUMO

Secondary structure motifs and small protein domains can act as building blocks that are isolated and investigated to gain insights into protein global structure but can also modulate interactions with external partners. Most progress has been made in this field using synthetic peptides. Fragmentation of folded proteins by proteolytic enzymes that act preferentially on exposed and less structured sites can help to isolate shorter polypeptides with preserved secondary and tertiary structures that mimic the original protein architecture. Such molecules can be used as probes for structural studies and as tools for in vitro assays to select active fragments useful as agonists or antagonists of the original protein or as scaffolds for the design of more potent and selective ligands. This simple but effective proteolytic methodology has been successfully applied to determine antagonists of protein-protein interactions, allowing the identification of inhibitors with high efficacy and specificity. Here, we present several studies including the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes and phospholipase 1, believed to play a relevant role in the insulin resistance mechanism in phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-overexpressing tissues, the self-association of BCL10 caspase recruitment domain that mediates a protein oligomerization process responsible for NF-kappaB activation and the self-association of growth arrest and DNA damage-inducible factor 45 beta, a major player of the endogenous NF-kappaB-mediated resistance to apoptosis.


Assuntos
Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Peptídeos/química , Mapeamento de Interação de Proteínas/métodos , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Antígenos de Diferenciação/química , Antígenos de Diferenciação/metabolismo , Proteínas Reguladoras de Apoptose , Proteína 10 de Linfoma CCL de Células B , Sítios de Ligação , Dicroísmo Circular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Peptídeos/síntese química , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA