RESUMO
AIMS: The specificity of the lysine demalonylation substrates of the pharmaceutically attractive tumor promoter/suppressor SIRT5 is not comprehensively clarified. The present study re-analyses publicly available data and highlights potentially pharmaceutically interesting outcomes by the use of bioinformatics. MATERIALS AND METHODS: The interaction networks of SIRT5 malonylome from the wild-type and ob/ob (obese pre-diabetic type) mice were subjected to the pathway enrichment and gene function prediction analysis using GeneMania (3.5.2) application run under Cytoscape (3.9.1) environment. KEY FINDINGS: The analysis in the wild-type mice revealed the involvement of SIRT5 malonylome in Eukaryotic translation elongation (ETE; the nodes EF1A1, EEF2, EEF1D, and EEF1G), Amino acid and derivative metabolism (AADM), and Selenoamino acid metabolism (SAM). The tumor promoter/suppressor activity of SIRT5 is mediated through the tumor promoter substrates included in AADM (GLUD1, SHMT1, ACAT1), and the tumor suppressor substrates involved in AADM and SAM (ALDH9A1, BHMT, GNMT). Selen stimulates the expression of SIRT5 and other sirtuins. SIRT5 in turn regulates the selenocysteine synthesis, which creates a regulatory loop. The analysis of SIRT5 malonylome in pre-diabetic ob/ob mice identifies the mTORC1 pathway as a mechanism, which facilitates SIRT5 functions. The comparison of the outcomes of SIRT5 malonylome, succinylome, and glutarylome analysis disclosed several differences. SIGNIFICANCE: The analysis showed additional aspects of SIRT5 malonylome functions besides the control of glucose metabolism. It defined several unique substrates and pathways, and it showed differences compared to other enzymatic activities of SIRT5, which could be used for pharmaceutical benefits.
Assuntos
Estado Pré-Diabético , Sirtuínas , Camundongos , Animais , Lisina/metabolismo , Glicólise/genética , Processamento de Proteína Pós-Traducional , Carcinógenos , Sirtuínas/metabolismoRESUMO
SIRT3 is a protein lysine deacetylase with a prominent role in the maintenance of mitochondrial integrity, which is a vulnerable target in many diseases. Intriguingly, cellular aging is reversible just by SIRT3 overexpression, which raises many questions about the role of SIRT3 in the molecular anti-aging mechanisms. Therefore, functions of SIRT3 were analyzed through the interaction network of 407 substrates collected by data mining. Results of the pathway enrichment and gene function prediction confirmed functions in the primary metabolism and mitochondrial ATP production. However, it also suggested involvement in thermogenesis, brain-related neurodegenerative diseases Alzheimer's (AD), Parkinson's, Huntington's disease (HD), and non-alcoholic fatty liver disease. The protein node prioritization analysis identified subunits of the complex I of the mitochondrial respiratory chain (MRC) as the nodes with the main regulatory effect within the entire interaction network. Additional high-ranked nodes were succinate dehydrogenase subunit B (SDHB), complex II, and ATP5F1, complex V of MRC. The analysis supports existence of the NADH/NAD+ driven regulatory feedback loop between SIRT3, complex I (MRC), and acetyl-CoA synthetases, and existence of the nuclear substrates of SIRT3. Unexplored functions of SIRT3 substrates such as LMNA and LMNB; HIF-1a, p53, DNA-PK, and PARK7 are highlighted for further scientific advances. SIRT3 acts as a repressor of BACE1 through the SIRT3-LKB1-AMPK-CREB-PGC1A-PPARG-BACE1 (SIRT3-BACE1), which functions are fitted the best by the Circadian Clock pathway. It forms a new working hypothesis as the therapeutical target for AD treatment. Other important pathways linked to SIRT3 activity are highlighted for therapeutical interventions.
Assuntos
Sirtuína 3 , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
Recently, all applications of BACE1 inhibitors failed as therapeutical targets for Alzheimer´s disease (AD) due to severe side effects. Therefore, alternative ways for treatment development are a hot research topic. The present analysis investigates BACE1 protein-protein interaction networks and attempts to solve the absence of complete knowledge about pathways involving BACE1. A bioinformatics analysis matched the functions of the non-substrate interaction network with Voltage-gated potassium channels, which also appear as top priority protein nodes. Targeting BACE1 interactions with PS1 and GGA-s, blocking of BACE1 access to APP by BRI3 and RTN-s, activation of Wnt signaling and upregulation of ß-catenin, and brain delivery of the extracellular domain of p75NTR, are the main alternatives to the use of BACE 1 inhibitors highlighted by the analysis. The pathway enrichment analysis also emphasized substrates and substrate candidates with essential biological functions, which cleavage must remain controlled. They include ephrin receptors, ROBO1, ROBO2, CNTN-s, CASPR-s, CD147, CypB, TTR, APLP1/APLP2, NRXN-s, and PTPR-s. The analysis of the interaction subnetwork of BACE1 functionally related to inflammation identified a connection to three cardiomyopathies, which supports the hypothesis of the common molecular mechanisms with AD. A lot of potential shows the regulation of BACE1 activity through post-translational modifications. The interaction network of BACE1 and its phosphorylation enzyme CSNK1D functionally match the Circadian clock, p53, and Hedgehog signaling pathways. The regulation of BACE1 glycosylation could be achieved through N-acetylglucosamine transferases, α-(1â6)-fucosyltransferase, ß-galactoside α-(2â6)-sialyltransferases, galactosyltransferases, and mannosidases suggested by the interaction network analysis of BACE1-MGAT3. The present analysis proposes possibilities for the alternative control of AD pathology.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/uso terapêutico , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Proteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Tripeptidyl peptidase II (TPPII) is primarily considered a house-keeping exopeptidase, which contributes to the functions of the ubiquitin-proteasome system by the maintenance of the cellular amino acid homeostasis. Although functionally well-characterised in vitro and using the mammalian cell models, less is known about the molecular mechanisms of its involvement in the signalling and metabolic pathways, which mediate its cellular functions. The present protein-protein interaction network analysis identified these mechanisms involved in the adaptive and innate immunity, the metabolism of the glucose, cancer cell growth, apoptosis, cell cycle and DNA damage responses. The interaction network constructed based on the publicly available protein-protein interaction data was extended by the application GeneMania, which was further used for the pathway enrichment, the protein function prediction and the protein node prioritisation analysis. The analysis suggested that the molecular mechanisms linked to the adaptive and innate immunity (ID, Kit receptor, BCR, IL-2 and G-CSF signalling; the regulation of NFκB), the aerobic glycolysis (ID and IL-2 signalling), tumorigenesis (TGF-ß and p53 signalling; the top priority nodes MAPKs, mTOR regulation), diabetes (Kit receptor signalling; the top priority node GSK3ß) and neurodegeneration (the control of mTOR and Aß peptide degradation) are controlling the resulting TPPII interaction network. The uncharacterized interactions with two lung cancer suppressors (DOK3, DENND2D), a protein involved in the increased risk of the lung cancer in smokers (CYP1A1) and a protein implicated in asthmatic reactions (CHIA) suggest potential roles of TPPII in the lung cancer pathology. The interactions with methyltransferase CARNMT1, which modifies di- and tripeptides and the xenobiotic processing enzyme CYP1A1, are additional candidates for the breakthrough in new functions discovery of TPPII.
RESUMO
PIN7 is a protein-protein interaction network of seven pleiotropic proteins (TPPII, CDK2, MYBBP1A, p53, SIRT6, SIRT7, and CD147) with proposed multiple functions in the aging and age-related diseases including cancer and neurodegeneration. Since the animal and cellular models with downregulated or knockout TPPII, p53, SIRT6, SIRT7, and MYBBP1A expression levels demonstrate similar age-related phenotype features, the interaction network was subjected to further investigation. For the identification of the main molecular mechanisms enabling the functions of the interaction network, PIN7 was subjected to the pathway enrichment, protein function prediction, and the protein node prioritization analysis using Cytoscape software and its applications GeneMania, ClusterOne, and Cyto-hubba. The study identified the p53 signaling pathway as the most dominant mediator of PIN7 effect. The top-ranked protein nodes of PIN7 extended by GeneMania application belong to the group of histone acetyltransferases and histone deacetylases. These enzymes are involved in the reverse epigenetic regulation mechanisms linked to the regulation of PTK2, NFκB, and p53 signaling interaction subnetworks of the extended PIN7. The analysis emphasized the role of PTK2 signaling, which functions upstream of the p53 signaling pathway, and its interaction network includes all top rank protein nodes of the extended PIN7 and all members of the sirtuin family (SIRT1-SIRT7). Further, the analysis suggests the involvement of molecular mechanisms related to metastatic cancer (prostate cancer, small cell lung cancer), hemostasis, the regulation of the thyroid hormones, and the cell cycle G1/S checkpoint. The additional data-mining analysis shows that the protein interaction network MYBBP1A-p53-TPPII-SIRT6-CD147 controls the Warburg effect, and MYBBP1A-p53-TPPII-SIRT7-CD147 influences mTOR signaling and autophagy. The proposed insights into the molecular mechanisms of aging and age-related diseases could be valuable for the discovery of new controlling interaction clusters, which could contribute to the development of the multitarget therapeutical strategies.
Assuntos
Mapas de Interação de Proteínas , Animais , Basigina , Quinase 2 Dependente de Ciclina , Proteínas de Ligação a DNA , Epigênese Genética , Células HEK293 , Humanos , Proteínas de Ligação a RNA , Transdução de Sinais , Sirtuínas , Fatores de Transcrição , Proteína Supressora de Tumor p53RESUMO
The present review summarizes the knowledge about a protein-interaction network, which includes proteins with significant functions in the mechanisms of aging and age-related diseases. All the detected interacting proteins TPPII, p53, MYBBP1A, CDK2 and SIRT7, SIRT6, and CD147 are suitable for the development of antitumor therapeutics and treatments for diseases of aging. TPPII and SIRT6 directly affect glucose metabolism which drive malignant growth. In addition, SIRT6 activators are attractive candidates for Alzheimer's disease (AD) due to the protection effect of SIRT6 overexpression from DNA damage. TPPII activity exhibits a decreasing effect on mTOR signaling, and its requirement for the degradation of Aß peptides in the human fibroblasts suggests that it has dual functions in tumorigenesis and AD-related pathology. Likewise, the direct promotion of the invasiveness of breast epithelial cells and the contribution to the Aß degradation by stimulating the matrix metalloproteinases production suggest a double functional role for CD147. An association of the partial portion of cellular CD147 to γ-secretase further supports the functional relation to AD pathology. The animal and cellular models with downregulated or knockout TPPII, p53, SIRT6, SIRT7, and MYBBP1A expression levels illustrate similar functions of the interacting proteins. They demonstrate similar effects on the length of life span, premature aging, and lipid metabolism. The presented protein-interaction network is relevant to the discoveries of the mechanisms of tumorigenesis, aging, and neurodegeneration.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/patologia , Animais , Transformação Celular Neoplásica/patologia , HumanosRESUMO
Novel protein-protein interactions of TPPII, SIRT7, and p53 were detected by co-immunoprecipitation using both HeLa cell lysates and the cytoplasmic fraction prepared by fractionation of mouse liver tissue. The interactions were further verified in vivo by in situ proximity ligation assay (PLA) within control HEK293 cells transformed with empty vector, highactTPPII HEK293 cells over-expressing murine TPPII displaying high specific enzymatic activity and in lowactTPPII HEK293 cells over-expressing human TPPII having low specific activity of the enzyme. Besides an abundant cytoplasmic localization of TPPII-p53 interaction signal, the nuclear interactions were also demonstrated. The cytoplasmic interactions were likewise detected between TPPII and SIRT7 in control HEK293 and lowactTPPII HEK293 cells. The interactions of SIRT7 with p53 were confirmed in three HEK293 cell transformants as well. The cytoplasmic occurrence of SIRT7 protein was demonstrated by immunofluorescence, when both nucleolar and cytoplasmic signals were identified within HEK293 cells and primary human fibroblasts. The unique cytoplasmic localization of SIRT7 protein was discussed based on an epitope specificity of N-terminus specific SIRT7 antibodies utilized in the present study compared with C-terminus specific antibodies previously used for nuclear detection of SIRT7 by other authors. The epitope sequence of N-terminal antibodies is occurring in all three splicing variants of SIRT7 compared to the epitope of C-terminal antibody, which is specific exclusively to the splicing variant 1. The cytoplasmic localization of p53 detected by immunofluorescence supported the results from its interactions with TPPII and SIRT7 observed by in situ PLA within model cells. Novel interactions of TPPII, p53, and SIRT7 presented in this study might contribute to the knowledge of the regulatory effects of these proteins on apoptotic pathways and to the understanding mechanisms of aging and lifespan regulation.
Assuntos
Aminopeptidases/metabolismo , Apoptose/fisiologia , Senescência Celular/fisiologia , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Serina Endopeptidases/metabolismo , Sirtuínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Aminopeptidases/genética , Animais , Linhagem Celular Tumoral , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Mapas de Interação de Proteínas , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/genéticaRESUMO
Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein-protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein-protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein-protein interaction network of these proteins.
Assuntos
Aminopeptidases/biossíntese , Quinase 2 Dependente de Ciclina/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/biossíntese , Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/biossíntese , Proteínas de Transporte Nucleocitoplasmático/biossíntese , Biossíntese de Proteínas/fisiologia , Serina Endopeptidases/biossíntese , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/genética , Anoikis/efeitos dos fármacos , Anoikis/fisiologia , Quinase 2 Dependente de Ciclina/genética , Proteínas de Ligação a DNA , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Indóis/farmacologia , Células K562 , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação a RNA , Serina Endopeptidases/genética , Fatores de TranscriçãoRESUMO
BACKGROUND: A key player in the development of Alzheimer's disease (AD) is the gamma-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. gamma-Secretase is crucial for the generation of the neurotoxic amyloid beta-peptide (Abeta) but also takes part in the processing of many other substrates. In cell lines, active gamma-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active gamma-secretase in the affected organ of AD, namely the brain. PRINCIPAL FINDINGS: We show by subcellular fractionation of rat brain that high gamma-secretase activity, as assessed by production of Abeta40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Abeta40 production and contained high amounts of the gamma-secretase components. Further purification of the synaptic vesicles verified the presence of the gamma-secretase components in these compartments. The localization of an active gamma-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated gamma-secretase inhibitor together with confocal microscopy. SIGNIFICANCE: The information about the subcellular localization of gamma-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active gamma-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Encéfalo/enzimologia , Endossomos/enzimologia , Sinapses/enzimologia , Animais , Masculino , Microscopia Confocal , Microscopia Eletrônica , Ratos , Ratos Sprague-DawleyRESUMO
Gamma-secretase activity has been extensively investigated due to its role in Alzheimer's disease. Here, we studied the association of CD147, a transmembrane glycoprotein belonging to the immunoglobulin family, with gamma-secretase and its expression in Alzheimer's disease and control tissues. Subcellular fractionation of postmitochondrial supernatant from rat brain on step iodixanol gradient in combination with co-immunoprecipitation using an anti-nicastrin antibody showed association of limited amount of CD147 to gamma-secretase. By immunoblotting of postnuclear pellets from Alzheimer's disease and control human brain tissues we showed that CD147 with molecular weight 75 kDa is upregulated in frontal cortex and thalamus of the Alzheimer's disease brains. Immunohistochemistry of brain tissues from Alzheimer's disease and control revealed specific upregulation of CD147 in neurons, axons and capillaries of Alzheimer's disease frontal cortex and thalamus. The effect of presenilin-1 and -2, which are the catalytic subunits of gamma-secretase, on CD147 expression and subcellular localization was analyzed by confocal microscopy in combination with flow cytometry and showed that PS2 affected the subcellular localization of CD147 in mouse embryonic fibroblast cells. We suggest that a small fraction of CD147 present in the brain is associated with the gamma-secretase, and can be involved in mechanisms dysregulated in Alzheimer's disease brain.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Basigina/metabolismo , Encéfalo/metabolismo , Presenilina-2/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/fisiopatologia , Compartimento Celular/fisiologia , Células Endoteliais/metabolismo , Feminino , Lobo Frontal/metabolismo , Células HeLa , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Transporte Proteico/fisiologia , Ratos , Frações Subcelulares/metabolismo , Tálamo/metabolismo , Regulação para Cima/fisiologiaRESUMO
The colonization process of tomato roots inoculated separately or/and simultaneously by a pathogenic Fusarium oxysporum f. sp. lycopersici strain Fol8 and the protective F. oxysporum strain Fo47, genetically tagged with the red and green fluorescent protein genes, respectively, was studied in a hydroponic culture. Plants were coinoculated with Fol8 and Fo47 at two conidial concentration ratios of 1/1 and 1/100, in which biological control was not effective or effective, respectively. First observation of fungi on root was possible 48 h after inoculation at a high inoculum level and 5 days post inoculation at the lower concentration of inoculum. The pattern of root colonization was similar for both strains with the initial development of hyphal network on the upper part of taproot, followed by the growth of hyphae towards the elongation zone, lateral roots and root apices. Finally, the whole elongation zone and root apex were invaded by both strains but no specific infection sites were observed. When coinoculated, both strains could grow very closely or even at the same spot on the root surface. At the nonprotective ratio, Fol8 was the successful colonizer, but application of Fo47 at a concentration 100 times >Fol8 delayed vessel colonization by the pathogen.
Assuntos
Fusarium/crescimento & desenvolvimento , Hidroponia , Raízes de Plantas/microbiologia , Solanum lycopersicum/microbiologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hifas/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Coloração e Rotulagem/métodos , Fatores de Tempo , Proteína Vermelha FluorescenteRESUMO
To investigate functional differences in the recognition and response mechanisms of conifer roots to fungi with different trophic strategies, Pinus sylvestris L. was challenged with a saprotrophic fungus Trichoderma aureoviride Rifai. The results were compared with separate studies investigating pine interactions with a pathogen, Heterobasidion annosum (Fr.) Bref. sensu stricto and an ectomycorrhizal symbiont, Laccaria bicolor Maire (Orton). Global changes in the expression of 2109 conifer genes were assayed 1, 5 and 15 days after inoculation. Gene expression data from a cDNA microarray were analyzed by the 2-interconnected mixed linear model statistical approach. The total number of genes differentially expressed compared with the uninfected control was similar after challenge with the pathogen and the ectomycorrhizal symbiont, but the number of differentially expressed genes increased over time for H. annosum, and decreased for L. bicolor. Inoculation of pine roots with T. aureoviride resulted overall in a much lower number of genes with changed transcript levels compared with inoculation with H. annosum or L. bicolor. Functional classification of the differentially expressed genes revealed that the ectomycorrhizal fungus triggered transient induction of defence-related genes. The response and induction of defence against the pathogen was delayed and the magnitude increased over time. Thus, there were specific transcriptional responses depending on whether the conifer roots were challenged with mutualistic, saprotrophic or pathogenic fungi. This suggests that pine trees are able to recognize diverse fungal species and specifically distinguish whether they are pathogenic, neutral or beneficial microbial agents.
Assuntos
Basidiomycota/patogenicidade , Pinus sylvestris/genética , Pinus sylvestris/microbiologia , Doenças das Plantas/microbiologia , Plântula/microbiologia , Transcrição Gênica , Trichoderma/patogenicidade , Comunicação Celular , Microscopia Eletrônica de Varredura , Pinus sylvestris/fisiologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/ultraestrutura , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/metabolismo , Trichoderma/ultraestruturaRESUMO
In soil, fungal colonization of plant roots has been traditionally studied by indirect methods such as microbial isolation that do not enable direct observation of infection sites or of interactions between fungal pathogens and their antagonists. Confocal laser scanning microscopy was used to visualize the colonization of tomato roots in heat-treated soil and to observe the interactions between a nonpathogenic strain, Fo47, and a pathogenic strain, Fol8, inoculated onto tomato roots in soil. When inoculated separately, both fungi colonized the entire root surface, with the exception of the apical zone. When both strains were introduced together, they both colonized the root surface and were observed at the same locations. When Fo47 was introduced at a higher concentration than Fol8, it colonized much of the root surface, but hyphae of Fol8 could still be observed at the same location on the root. There was no exclusion of the pathogenic strain by the presence of the nonpathogenic strain. These results are not consistent with the hypothesis that specific infection sites exist on the root for Fusarium oxysporum and instead support the hypothesis that competition occurs for nutrients rather than for infection sites.
Assuntos
Fusarium/patogenicidade , Solanum lycopersicum/microbiologia , Fusarium/crescimento & desenvolvimento , Solanum lycopersicum/crescimento & desenvolvimento , Microscopia Confocal , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Microbiologia do Solo , Especificidade da Espécie , VirulênciaRESUMO
A new family of antimicrobial peptide homologues termed Sp-Amp has been discovered in Pinus sylvestris (Scots pine). This is the first report of such proteins to be characterized in a conifer species. Sp-AMP1 was identified in a substructured cDNA library of root tissue infected with the root rot fungus Heterobasidion annosum and encodes a mature peptide of 79 amino acid residues. Three additional members of the Sp-AMP family (Sp-AMPs 2-4) encode cysteine-rich proteins of 105 amino acids, each containing an N-terminal region with a probable cleavage signal sequence. Northern analysis confirmed that Sp-AMP expression is elevated in Scots pine roots upon infection with H. annosum. These peptides share 64% amino acid identity with a mature protein from Macadamia integrifolia (MiAMP1), which allowed us to build a homology model for preliminary analysis. Southern analyses further confirmed that several copies of the gene are present in the Scots pine genome. The potential significance of Sp-AMP in the H. annosum-conifer pathosystem is discussed.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Basidiomycota , Pinus sylvestris/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Sequência de Bases , Dados de Sequência Molecular , Micoses/microbiologia , Pinus sylvestris/microbiologia , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Homologia de Sequência de AminoácidosRESUMO
pAn-DsRed2 vector was constructed for constitutive cytoplasmic expression of the red fluorescent protein (DsRed2) under control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter from Aspergillus nidulans. DsRed2-transformation of two Fusarium oxysporum f. sp. lycopersici strains pathogenic against tomato host resulted in bright red cytoplamic fluorescence of the fungus. The transformants were screened based on the hygromycin B resistance, brightness, stability and rate of appearance of the DsRed2 fluorescence. The transormed fungi were growing normally and their pathogenicity did not change after transformation procedure. The function of novel DsRed2 marker was verified by fluorescence microscopy of the infected tomato seedlings. The results indicate that DsRed2 can be used as a efficient novel reporter gene for monitoring of the F. oxysporum within the host tissues.
Assuntos
Fusarium/genética , Fusarium/metabolismo , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Aspergillus nidulans/enzimologia , Clonagem Molecular , Fluorescência , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Genes , Glicerol-3-Fosfato Desidrogenase (NAD+) , Glicerolfosfato Desidrogenase/genética , Solanum lycopersicum/microbiologia , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Transformação Genética , Proteína Vermelha FluorescenteRESUMO
A biosensor based on the surface plasmon resonance (SPR) principle was used for kinetic analysis of lectin interactions with different immobilized saccharide structures. A novel affinity ligands beta-D-glycopyranosylmethylamines derived from common D-aldohexoses linked to the carboxymethyl dextran layer of the SPR sensor surface served for interactions with a wide range of lectins. The method of preparation and use of the beta-D-mannopyranosyl glycosylated sensor surface was described. The results of affinity analysis of lectin-ligand interactions were evaluated and compared with data obtained from measurements using commercially available p-aminophenyl alpha-D-glycopyranosides. Possible applications and advantages of C- and O-glycosylated SPR biosensors are discussed.