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6-methyladenine (6mA) is fairly abundant in nuclear DNA of basal fungi, ciliates and green algae. In these organisms, 6mA is maintained near transcription start sites in ApT context by a parental-strand instruction dependent maintenance methyltransferase and is positively associated with transcription. In animals and plants, 6mA levels are high only in organellar DNA. The 6mA levels in nuclear DNA are very low. They are attributable to nucleotide salvage and the activity of otherwise mitochondrial METTL4, and may be considered as a price that cells pay for adenine methylation in RNA and/or organellar DNA. Cells minimize this price by sanitizing dNTP pools to limit 6mA incorporation, and by converting 6mA that has been incorporated into DNA back to adenine. Hence, 6mA in nuclear DNA should be described as an epigenetic mark only in basal fungi, ciliates and green algae, but not in animals and plants.
Assuntos
Metilação de DNA , Eucariotos , Adenina , Animais , DNA/metabolismo , Dano ao DNA , Eucariotos/genéticaRESUMO
Diabetic retinopathy (DR) is a severe disease with a growing number of afflicted patients, which places a heavy burden on society, both socially and financially. While there are treatments available, they are not always effective and are usually administered when the disease is already at a developed stage with visible clinical manifestation. However, homeostasis at a molecular level is disrupted before visible signs of the disease are evident. Thus, there has been a constant search for effective biomarkers that could signal the onset of DR. There is evidence that early detection and prompt disease control are effective in preventing or slowing DR progression. Here, we review some of the molecular changes that occur before clinical manifestations are observable. As a possible new biomarker, we focus on retinol binding protein 3 (RBP3). We argue that it displays unique features that make it a very good biomarker for non-invasive, early-stage DR detection. Linking chemistry to biological function and focusing on new developments in eye imaging and two-photon technology, we describe a new potential diagnostic tool that would allow rapid and effective quantification of RBP3 in the retina. Moreover, this tool would also be useful in the future to monitor therapeutic effectiveness if levels of RBP3 are elevated by DR treatments.
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Diabetes Mellitus , Retinopatia Diabética , Proteínas de Ligação ao Retinol , Humanos , Biomarcadores/metabolismo , Diabetes Mellitus/metabolismo , Retinopatia Diabética/metabolismo , Diagnóstico por Imagem , Retina/metabolismo , Retinoides/metabolismoRESUMO
Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of conifer trees. Here, we identified multiple novel members in the western white pine (Pinus monticola) PmPR10 family by bioinformatic mining available transcriptomic data. Phylogenetic analysis of protein sequences revealed four PR10 and two PR10-like clusters with a high synteny across different species of five-needle pines. Of 10 PmPR10 genes, PmPR10-3.1 was selected and expressed in Escherichia coli. The purified recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens Cronartium ribicola, Phoma exigua, and Phoma argillacea by in-vitro anti-fungal analysis. Genetic variation analysis detected a total of 21 single nucleotide polymorphisms (SNPs) within PmPR10-3.1 in a collection of P. monticola seed families. A nonsynonymous SNP (t178g) showed significant association with relative levels of quantitative disease resistance (QDR), explaining about 8.7% of phenotypic variation as the peak value across all SNPs. Our results provide valuable insight into the genetic architecture underlying P. monticola QDR and imply that PmPR10-3.1 may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.
Assuntos
Basidiomycota , Resistência à Doença , Pinus , Doenças das Plantas , Basidiomycota/patogenicidade , Resistência à Doença/genética , Humanos , Phoma/patogenicidade , Filogenia , Pinus/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The type II and type V CRISPR effector nucleases Cas9 and Cpf1 are "universal" DNA endonucleases, which can be programmed by an appropriate crRNA or sgRNA strand to cleave almost any DNA duplex at a preselected position (constrained only by short, so-called PAMs). In this review, we briefly introduce CRISPR bacterial adaptive immunity as the biological context in which Cas9 and Cpf1 proteins operate, and then present the structural insights that have been obtained in the last two or three years that illustrate the mode of operation of these proteins. We describe the R-loop structures at the core of the Cas9 and Cpf1 complexes, and the structure of the 5'- or 3'-handles that help anchor the nucleic acid complexes to the proteins in a manner that is independent of the target sequence. Next, we describe the molecular architecture of the Cas9 and Cpf1 proteins. We illustrate how Cas9 and Cpf1 proteins scan double stranded DNA for so-called protospacer associated motifs (PAMs), we explain how the phosphate loop (PLL) and basic helix (BH) promote the separation of target and non-target DNA strands and the formation of hybrids between crRNA or sgRNA and the target strand of DNA. We also describe the current understanding of the catalytic mechanisms of RuvC and HNH domains, and a possible, but still very uncertain catalytic role of the Nuc domain. At the end of the review, we briefly summarize key developments that have initiated the field of genomic engineering using Cas9 or Cpf1 nucleases.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Bactérias/genética , Proteínas de Bactérias/química , Conformação ProteicaRESUMO
Two-photon excitation fluorescence (TPEF) is emerging as a powerful imaging technique with superior penetration power in scattering media, allowing for functional imaging of biological tissues at a subcellular level. TPEF is commonly used in cancer diagnostics, as it enables the direct observation of metabolism within living cells. The technique is now widely used in various medical fields, including ophthalmology. The eye is a complex and delicate organ with multiple layers of different cell types and tissues. Although this structure is ideal for visual perception, it generates aberrations in TPEF eye imaging. However, adaptive optics can now compensate for these aberrations, allowing for improved imaging of the eyes of animal models for human diseases. The eye is naturally built to filter out harmful wavelengths, but these wavelengths can be mimicked and thereby utilized in diagnostics via two-photon (2Ph) excitation. Recent advances in laser-source manufacturing have made it possible to minimize the exposure of in vivo measurements within safety, while achieving sufficient signals to detect for functional images, making TPEF a viable option for human application. This review explores recent advances in wavefront-distortion correction in animal models and the safety of use of TPEF on human subjects, both of which make TPEF a potentially powerful tool for ophthalmological diagnostics.
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Effectors of the Rab small GTPases are large multi-domain proteins which have proved difficult to express in soluble form in Escherichia coli. Generally, effectors are recruited to a distinct subcellular compartment by active (GTP-bound) Rabs, which are linked to membranes by one or two prenylated Cys residues at their C-termini. Following recruitment via their Rab-binding domain (RBD), effectors carry out various aspects of vesicle formation, transport, tethering and fusion through their other domains. Previously, successful purification of the RUN-PLAT tandem domains (residues 683-1061) of the 1263-residue Rab6-interacting protein 1 (R6IP1) required co-expression with Rab6, as attempts to solubly express the effector alone were unsuccessful. R6IP1 is also known as DENN domain-containing protein 5 (DENND5) and is expressed as two isoforms, R6IP1A/B (DENND5A/B), which differ by 24 amino acids at the N-terminus. Here, a deletion in R6IP1 was engineered to enable soluble expression and to improve the quality of the crystals grown in complex with Rab6. A large 23-residue loop linking two α-helices in the RUN1 domain was removed and replaced with a short linker. This loop resides on the opposite face to the Rab6-binding site and is not conserved in the RUN-domain family. In contrast to wild-type R6IP1-Rab6 crystals, which took several weeks to grow to full size, the engineered R6IP1 (RPdel)-Rab6 crystals could be grown in a matter of days.
Assuntos
Proteínas rab de Ligação ao GTP/química , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Humanos , Camundongos , Dados de Sequência Molecular , Engenharia de Proteínas , Alinhamento de SequênciaRESUMO
Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, widely known as St. John's wort. Hypericin has been attracting a growing attention of the pharmaceutical industry because of its potential application in various therapies, including the treatment of depression. In vivo, hypericin is synthesized by dimerization of emodin in a complicated multistep reaction that is reportedly catalyzed by a small (17.8kDa) protein, Hyp-1. Based on relatively low sequence similarity ( approximately 50%), Hyp-1 has been tentatively classified as a plant PR-10 (pathogenesis-related class 10) protein. Members of the PR-10 family are ubiquitous plant proteins associated with stress control and tissue differentiation but with no clearly understood molecular mechanism. They have, however, a well-defined folding canon, consisting of an extended antiparallel beta-sheet wrapped around a C-terminal alpha-helix, enclosing in the protein interior a huge cavity, in which various hydrophobic ligands can be bound. Apart from Hyp-1, only two other PR-10 members have been found to possess enzymatic activity (S-norcoclaurine synthase and TcmN aromatase/cyclase). In this paper, we report a high-resolution crystal structure of Hyp-1, confirming that it indeed has a PR-10 fold. The protein binds multiple polyethylene glycol molecules, some of which occupy the hydrophobic cavity. The crystallographic model illustrates a high degree of conformational adaptability of both interacting partners for efficient binding. We have been unable, however, to dimerize emodin to hypericin using Hyp-1 as biocatalyst. This puzzling result does not have a clear explanation at this time.
Assuntos
Antidepressivos/metabolismo , Hypericum/química , Modelos Moleculares , Perileno/análogos & derivados , Proteínas de Plantas/química , Conformação Proteica , Antracenos , Cristalização , Dimerização , Emodina/química , Estrutura Molecular , Perileno/metabolismo , Proteínas de Plantas/metabolismoRESUMO
The small GTPase Rab6 regulates vesicle trafficking at the level of Golgi. Recently, the crystal structures of Rab6 in complexes with two unrelated effectors have been determined. The structure of Rab6a-GTP in complex with a 378-residue internal fragment of the effector Rab6IP1 (Rab6-interacting protein 1) has been solved. In addition, the structure of Rab6 with the golgin, GCC185, has also been determined. In both complexes, two alpha-helices from the effector mediate binding to switch I, switch II and the interswitch region of Rab6. Comparisons of the complexes reveal significant conformational changes in the conserved hydrophobic triad of Rab6. Thus conformational flexibility in the triad mediates recognition of compositionally distinct alpha-helical coiled coils, providing a rationale for the promiscuity of Rab6 in effector recruitment.
Assuntos
Conformação Proteica , Proteínas rab de Ligação ao GTP/química , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com-RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.
Assuntos
Bacteriófago mu/genética , RNA Complementar/química , Proteínas Virais/química , Dedos de Zinco , Pareamento de Bases , Sítios de Ligação , DNA/metabolismo , Genes Virais , Haemophilus , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/genética , Técnica de Seleção de Aptâmeros , Solventes , Transcrição GênicaRESUMO
According to a debated hypothesis, the biosynthesis from emodin of the medicinally important natural compound hypericin is catalyzed in St John's wort (Hypericum perforatum) by the phenolic oxidative-coupling protein Hyp-1. Recombinant St John's wort Hyp-1 has been overexpressed in Escherichia coli and obtained in single-crystal form. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell parameters a = 37.5, b = 76.7, c = 119.8 A, contain two protein molecules in the asymmetric unit and diffract X-rays to 1.73 A resolution.
Assuntos
Hypericum/química , Perileno/análogos & derivados , Proteínas de Plantas/química , Sequência de Aminoácidos , Antracenos , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Perileno/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif (RRM) domain. We have determined the crystal structure of the human Acinus RRM domain (AcRRM) at 1.65 Å resolution. It shows a classical four-stranded antiparallel ß-sheet fold with two flanking α-helices and an additional, non-classical α-helix at the C-terminus, which harbors the caspase-3 target sequence that is cleaved during Acinus activation. In the structure, the C-terminal α-helix partially occludes the potential ligand binding surface of the ß-sheet and hypothetically shields it from non-sequence specific interactions with RNA. Based on the comparison with other RRM-RNA complex structures, it is likely that the C-terminal α-helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinus.
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Noroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV) NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction) into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease-substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P') side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein (NS6 1', NS6 2', NS6 4'). We also determined the structure of a chimeric extended NS6 protease in which the P4-P4' sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4' 2|3).The constructs NS6 1' and NS6 2' yielded crystals that diffracted anisotropically. We found that, although the uncorrected data could be phased by molecular replacement, refinement of the structures stalled unless the data were ellipsoidally truncated and corrected with anisotropic B-factors. These corrections significantly improved phasing by molecular replacement and subsequent refinement.The refined structures of all four extended NS6 proteases are very similar in structure to the mature MNV NS6-and in one case reveal additional details of a surface loop. Although the packing arrangement observed showed some similarities to those observed in the adventitious protease-product crystals reported previously, in no case were specific protease-substrate interactions observed.
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Physical, chemical and biological stress factors, such as microbial infection, upregulate the transcription levels of a number of plant genes, coding for the so-called pathogenesis-related (PR) proteins. For PR proteins of class-10 (PR-10), the biological function remains unclear, despite two decades of scientific research. PR-10 proteins have a wide distribution throughout the plant kingdom and the class members share size and secondary structure organization. Throughout the years, we and other groups have determined the structures of a number of PR-10 proteins, both in the crystalline state by X-ray diffraction and in solution by NMR spectroscopy. Despite the accumulating structural information, our understanding of PR-10 function is still limited. PR-10 proteins are rather small (~ 160 amino acids) with a fold consisting of three α helices and seven antiparallel ß strands. These structural elements enclose a large hydrophobic cavity that is most probably the key to their functional relevance. Also, the outer surface of these proteins is of extreme interest, as epitopes from a PR-10 subclass cause allergic reactions in humans.
Assuntos
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump in E. coli for biophysical and structural analysis. We show that recombinant Ump is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.
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Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.
Assuntos
Calixarenos/química , Citocromos c/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citocromos c/química , Fenóis/química , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Solventes/química , Eletricidade EstáticaRESUMO
Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.
Assuntos
Complexo de Golgi/metabolismo , Nexinas de Classificação/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Complexo de Golgi/genética , Fatores de Troca do Nucleotídeo Guanina , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico/genética , Proteínas Recombinantes de Fusão , Nexinas de Classificação/química , Nexinas de Classificação/genética , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genéticaRESUMO
De acordo com as normas do Programa de Humanização no Pré-Natal e Nascimento, do Ministério da Saúde, a gestante deve realizar dois exames laboratoriais para detecção da sífilis. Objetivo: Avaliar oportunidades perdidas no rastreamento de sífilis gestacional e identificar fatores associados à não realização do teste Venereal Disease Research Laboratory (VDRL) no pré-natal. Métodos: Estudo de corte transversal realizado em maternidade do Sistema Único de Saúde da cidade do Recife, no Nordeste do Brasil. Foram estudadas 460 mulheres admitidas por término da gravidez e/ou abortamento, entre setembro e outubro de 2013, que realizaram ao menos uma consulta de pré-natal. Foram realizadas entrevistas e consulta ao cartão de pré-natal e prontuários. As mulheres que não realizaram pelo menos um VDLR no pré-natal (categoria de referência) foram comparadas com aquelas que realizaram. Nas informações colhidas nos 408 cartões da gestante, foi utilizada a regressão logística para identificar fatores associados com a não realização do rastreio. Resultados: Uma parte correspondente a 17,90% das mulheres não realizou o VDRL. Na análise multivariada, apresentaram maior chance de não realizar o VDRL as mulheres que: enfrentaram dificuldades para realizar o exame; realizaram a última consulta antes do último trimestre da gravidez; passaram por menos de seis consultas; realizaram pré-natal em unidade que não realizava agendamento das consultas subsequentes; tinham 19 anos ou menos de idade; tinham três ou mais gravidezes. Conclusão: Os resultados mostram que, apesar da elevada cobertura da atenção pré-natal, persiste uma baixa efetividade das ações de prevenção da sífilis congênita.
According to the norms issued by the Ministry of Health for the Prenatal and Birth Humanization Program, pregnant women should undergo two tests for syphilis detection. Objective: To evaluate missed opportunities for screening gestational syphilis and to identify factors associated with the missing application of the Venereal Disease Research Laboratory (VDRL) test during prenatal development. Methods: This cross-sectional study was undertaken in the maternity ward of a Unified Public Health System (SUS) hospital in the city of Recife in northeastern Brazil. We studied 460 women admitted for termination of pregnancy and/or abortion, between September and October 2013, who had at least one prenatal consultation. We conducted interviews and checked patients' prenatal care records and medical records. Women who did not take at least one VDRL test during prenatal development (reference category) were compared with those who did. Logistic regression was performed on the data collected from the 408 pregnancy records analyzed in order to identify factors associated with a failure to undergo syphilis screening. Results: 17.90% of the women in the sample did not take the VDRL test. In multivariate analysis, women who fit the following factors presented a greater chance of not having taken the VDRL test: facing difficulties in taking the test; attendance of the last prenatal consultation before the last trimester of pregnancy; attending less than six consultations; receiving prenatal care in hospital units which did not schedule subsequent exams; being 19 years of age or younger; having had three or more pregnancies. Conclusion: Results show that despite the high availability of prenatal care, actions aimed at preventing congenital syphilis still present low effectiveness.
Assuntos
Humanos , Feminino , Gravidez , Adulto , Cuidado Pré-Natal , Fatores de Risco , Sífilis Congênita/diagnóstico , Sífilis Congênita/epidemiologia , Triagem Neonatal/estatística & dados numéricos , Infecções Sexualmente TransmissíveisRESUMO
Plant pathogenesis-related (PR) proteins of class 10 are the only group among the 17 PR protein families that are intracellular and cytosolic. Sequence conservation and the wide distribution of PR-10 proteins throughout the plant kingdom are an indication of an indispensable function in plants, but their true biological role remains obscure. Crystal and solution structures for several homologues have shown a similar overall fold with a vast internal cavity which, together with structural similarities to the steroidogenic acute regulatory protein-related lipid transfer domain and cytokinin-specific binding proteins, strongly indicate a ligand-binding role for the PR-10 proteins. This article describes the structure of a complex between a classic PR-10 protein [Lupinus luteus (yellow lupine) PR-10 protein of subclass 2, LlPR-10.2B] and N,N'-diphenylurea, a synthetic cytokinin. Synthetic cytokinins have been shown in various bioassays to exhibit activity similar to that of natural cytokinins. The present 1.95 A resolution crystallographic model reveals four N,N'-diphenylurea molecules in the hydrophobic cavity of the protein and a degree of conformational changes accompanying ligand binding. The structural adaptability of LlPR-10.2B and its ability to bind different cytokinins suggest that this protein, and perhaps other PR-10 proteins as well, can act as a reservoir of cytokinin molecules in the aqueous environment of a plant cell.
Assuntos
Citocininas/farmacologia , Lupinus/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Plant pathogenesis-related (PR) proteins of class 10 (PR-10) are small and cytosolic. The main feature of their three-dimensional structure is a large cavity between a seven-stranded antiparallel beta-sheet and a long C-terminal alpha-helix. Although PR-10 proteins are abundant in plants, their physiological role remains unknown. Recent data have indicated ligand binding as their possible biological function. The article describes the structure of a complex between a classic PR-10 protein (yellow lupine LlPR-10.2B) and the plant hormone, trans-zeatin. Previously, trans-zeatin binding has been reported in a structurally related cytokinin-specific binding protein, which has a distant sequence relation with classic PR-10 proteins. In the present 1.35 A resolution crystallographic model, three perfectly ordered zeatin molecules are found in the binding cavity of the protein. The fact that three zeatin molecules are bound by the protein when only a fourfold molar excess of the ligand was used indicates an unusual type of affinity for this ligand and suggests that LlPR-10.2B, and perhaps other PR-10 proteins as well, acts as a reservoir of cytokinin molecules in the aqueous environment of the cell.
Assuntos
Citocininas/metabolismo , Lupinus/química , Reguladores de Crescimento de Plantas/química , Proteínas de Plantas/química , Estrutura Terciária de Proteína , Zeatina/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Citocininas/química , Dados de Sequência Molecular , Estrutura Molecular , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Zeatina/metabolismoRESUMO
Introdução: A histiocitose de células de Langerhans é um distúrbio proliferativo de células inflamatórias de etiologia desconhecida. É doença rara da faixa pediátrica. Objetivo: Realizar revisão de literatura sobre a histiocitose de células de Langerhans, focando as manifestações otorrinolaringológicas. Materiais e métodos: A metodologia utilizada foi consulta à base de dados on line MEDLINE, de 1966 a 2008, pesquisando a partir dos termos histiocitose de células de Langerhans, osso temporal e otorrinolaringologia. Revisão da Literatura: As manifestações em cabeça e pescoço são as mais frequentes e seu diagnóstico torna-se difícil uma vez que mimetiza outras doenças mais comuns vistas pelo otorrinolaringologista, como otite externa, mastoidite aguda e gengivite. A doença no osso temporal expressa-se como otorreia de repetição e granulomas de conduto auditivo externo ou retroauricular. A avaliação radiológica evidencia lesões líticas principalmente em calota craniana, mandíbula, osso temporal e costelas. O diagnóstico definitivo é feito por biópsia através dos achados histopatológicos e detecção pela imunohistoquímica do antígeno CD1a. A principal forma de tratamento é a quimioterapia e, em menor escala, radioterapia ou cirurgia. Conclusões: Manifestações otorrinolaringológicas devem ser suspeitadas por sintomas otológicos recorrentes e pela presença de tecido de granulação retroauricular ou de conduto auditivo externo. A biópsia com achados histológicos característicos e imunohistoquímica positiva para CD1a são diagnósticos. A quimioterapia pode ser o tratamento inicial na maioria dos casos ou adjuvante nas formas refratárias ou recorrentes.
Introduction: The Langerhans cell histiocytosis is an inflammatory cells proliferative disorder of unknown etiology. It is uncommon disease in children. Objective: To proceed with a literature review on Langerhans cell histiocytosis, with focus on the otorhinolaryngological complications. Materials and methods: The methodology used was advised based on online data from MEDLINE, between 1966 and 2008, with research of terms related to Langerhans cell histiocytosis, temporal bones and otorhinolaryngology. Literature Review: The manifestations in the head and neck are the most common ones and their diagnosis becomes difficult once it mimetizing other more common diseases the otorhinolaryngologist sees as external ear eczema, acute mastoiditis and gingivitis. The temporal bone disease manifests as recurrent otorrhea and external auditory meatus and retroauricular granulomas. The radiological evaluation confirms lytic lesions especially in the cranial cap, jaw, temporal bones and spines. The definitive diagnosis is made by biopsy through the histopathological discoveries and immunohistochemistry detection of the CD1a antigen. The main form of treatment is by chemotherapy and, in a lower scale, radiotherapy or surgery. Conclusions: he otorhinolaryngological manifestations must be suspected for recurrent otological symptoms and the presence of retroauricular granulation tissue or and the external auditory meatus. The biopsy with characteristic histological discoveries and immunohistochemistry positive for CD1a were diagnostic. The chemotherapy may be the initial treatment in most cases or secondary in refractory or recurrent forms.