RESUMEN
BACKGROUND: A subset of women with breast implants have reported a myriad of nonspecific systemic symptoms collectively termed systemic symptoms associated with breast implants (SSBI). SSBI symptoms are similar to manifestations associated with autoimmune and connective tissue disorders Breast tissue is rich in adipose cells, comprised of lipids. Insertion of an implant creates an oxidative environment leading to lipid oxidation. Oxylipins can influence immune responses and inflammatory processes. OBJECTIVES: This study explores the abundance of a spectrum of oxylipins in the peri-prosthetic tissue surrounding the breast implant. Since oxylipins are immunogenic, we sought to determine if they are associated with the SSBI subjects. We have also attempted to determine if the common manifestations exhibited by such subjects have any association with oxylipin abundance. METHODS: The study included 120 subjects divided in three cohorts. Forty-six subjects with breast implants exhibiting manifestation associated with SSBI, 29 in control cohort I subjects with breast implants not exhibiting manifestation associated with SSBI (non-SSBI) and 45 in control cohort II subjects without implants (normal tissue) were analyzed. Lipid extraction and oxylipin quantification was performed using LCMS. LC-MS/MS targeted analysis from the breast adipose tissue was performed. RESULTS: Of the fifteen oxylipins analyzed, four exhibited increased abundance in the SSBI cohort compared to the non-SSBI and normal cohorts. CONCLUSIONS: The study documents the association of the oxylipins with each manifestation reported by the subject. This study provides an objective assessment on the subjective questionnaire highlighting which symptoms could be more relevant than the others.
RESUMEN
Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.
Asunto(s)
Aminoácidos/genética , Transferasas Intramoleculares/genética , Proteínas de Plantas/genética , Triterpenos/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Avena/enzimología , Avena/genética , Avena/metabolismo , Secuencia Conservada/genética , Ciclización , Transferasas Intramoleculares/química , Transferasas Intramoleculares/metabolismo , Modelos Moleculares , Estructura Molecular , Mutación , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Dominios Proteicos , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Triterpenos/químicaRESUMEN
The Echinacea genus is exemplary of over 30 plant families that produce a set of bioactive amides, called alkamides. The Echinacea alkamides may be assembled from two distinct moieties, a branched-chain amine that is acylated with a novel polyunsaturated fatty acid. In this study we identified the potential enzymological source of the amine moiety as a pyridoxal phosphate-dependent decarboxylating enzyme that uses branched-chain amino acids as substrate. This identification was based on a correlative analysis of the transcriptomes and metabolomes of 36 different E. purpurea tissues and organs, which expressed distinct alkamide profiles. Although no correlation was found between the accumulation patterns of the alkamides and their putative metabolic precursors (i.e., fatty acids and branched-chain amino acids), isotope labeling analyses supported the transformation of valine and isoleucine to isobutylamine and 2-methylbutylamine as reactions of alkamide biosynthesis. Sequence homology identified the pyridoxal phosphate-dependent decarboxylase-like proteins in the translated proteome of E. purpurea. These sequences were prioritized for direct characterization by correlating their transcript levels with alkamide accumulation patterns in different organs and tissues, and this multi-pronged approach led to the identification and characterization of a branched-chain amino acid decarboxylase, which would appear to be responsible for generating the amine moieties of naturally occurring alkamides.
Asunto(s)
Amidas/metabolismo , Echinacea/genética , Echinacea/metabolismo , Metabolómica/métodos , Transcriptoma/genética , Biocatálisis , Ácidos Grasos/metabolismoRESUMEN
Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.
Asunto(s)
Agaricales/genética , Evolución Molecular , Genoma Fúngico , Madera/microbiología , Agaricales/enzimología , Agaricales/patogenicidad , Lignina/metabolismo , Filogenia , Análisis de Secuencia de ADNRESUMEN
This study investigates a mechanistic link of bacterial biofilm-mediated host-pathogen interaction leading to immunological complications associated with breast implant illness (BII). Over 10 million women worldwide have breast implants. In recent years, women have described a constellation of immunological symptoms believed to be related to their breast implants. We report that periprosthetic breast tissue of participants with symptoms associated with BII had increased abundance of biofilm and biofilm-derived oxylipin 10-HOME compared with participants with implants who are without symptoms (non-BII) and participants without implants. S. epidermidis biofilm was observed to be higher in the BII group compared with the non-BII group and the normal tissue group. Oxylipin 10-HOME was found to be immunogenically capable of polarizing naive CD4+ T cells with a resulting Th1 subtype in vitro and in vivo. Consistently, an abundance of CD4+Th1 subtype was observed in the periprosthetic breast tissue and blood of people in the BII group. Mice injected with 10-HOME also had increased Th1 subtype in their blood, akin to patients with BII, and demonstrated fatigue-like symptoms. The identification of an oxylipin-mediated mechanism of immune activation induced by local bacterial biofilm provides insight into the possible pathogenesis of the implant-associated immune symptoms of BII.
Asunto(s)
Implantes de Mama , Humanos , Femenino , Ratones , Animales , Implantes de Mama/efectos adversos , Implantes de Mama/microbiología , Oxilipinas , Biopelículas , InmunidadRESUMEN
Acetylenic specialized metabolites containing one or more carbon-carbon triple bonds are widespread, being found in fungi, vascular and lower plants, marine sponges and algae, and insects. Plants, moss, and most recently, insects, have been shown to employ an energetically difficult, sequential dehydrogenation mechanism for acetylenic bond formation. Here, we describe the cloning and heterologous expression in yeast of a linoleoyl 12-desaturase (acetylenase) and a bifunctional desaturase with Delta(12)-/Delta(14)-regiospecificity from the Pacific golden chanterelle. The acetylenase gene, which is the first identified from a fungus, is phylogenetically distinct from known plant and fungal desaturases. Together, the bifunctional desaturase and the acetylenase provide the enzymatic activities required to drive oleate through linoleate to crepenynate and the conjugated enyne (14Z)-dehydrocrepenynate, the branchpoint precursors to a major class of acetylenic natural products.
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Agaricus/enzimología , Ácido Graso Desaturasas/química , Ácidos Grasos/química , Proteínas Fúngicas/química , Agaricus/genética , Secuencia de Bases , Clonación Molecular , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Grasos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
The first synthesis of ganodermanontriol, a bioactive lanostane triterpene from the medicinal mushroom Ganoderma lucidum, has been achieved in 15.3% yield over nine steps, along with its three stereoisomeric triols and ganoderol A. The key steps leading to this family of isomers involve the reconstruction of the trisubstituted alkene by stereoselective and chemoselective phosphonate reactions and the formation of the unusual Δ7,9(11)-diene core by the mild acidic opening of a lanosterone-derived epoxide. Ganodermanontriol showed promising activity on the inhibition and proliferation of breast cancer cells. The effect of ganodermanontriol and its isomers on cell proliferation was assayed; IC50 values of 5.8 and 9.7 µM on breast cancer cell lines MCF-7 and MDA-MB-231, respectively, were found for ganodermanontriol.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Lanosterol/análogos & derivados , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Lanosterol/síntesis química , Lanosterol/química , Lanosterol/farmacología , Estructura Molecular , Reishi/química , EstereoisomerismoRESUMEN
Polyacetylenic natural products are a substantial class of often unstable compounds containing a unique carbon-carbon triple bond functionality, that are intriguing for their wide variety of biochemical and ecological functions, economic potential, and surprising mode of biosynthesis. Isotopic tracer experiments between 1960 and 1990 demonstrated that the majority of these compounds are derived from fatty acid and polyketide precursors. During the past decade, research into the metabolism of polyacetylenes has swiftly advanced, driven by the cloning of the first genes responsible for polyacetylene biosynthesis in plants, moss, fungi, and actinomycetes and the initial characterization of the gene products. The current state of knowledge of the biochemistry and molecular genetics of polyacetylenic secondary metabolic pathways will be presented together with an up-to-date survey of new terrestrial and marine natural products, their known biological activities, and a discussion of their likely metabolic origins.
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Hongos/metabolismo , Plantas/metabolismo , Poliinos/metabolismo , Actinobacteria/metabolismo , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Biología MolecularRESUMEN
The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Delta(12)-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structural domains. In vivo activity of the heterologously expressed enzyme favors C(18) substrates with nu+3 regioselectivity, where the site of desaturation is three carbons carboxy-distal to the reference position of a preexisting double bond (nu). Linoleate accumulated to levels in excess of 12% of the total fatty acids upon heterologous expression of P. chrysosporium FAD2 in Saccharomyces cerevisiae. In contrast to the behavior of the plant FAD2 enzymes, this oleate desaturase does not 12-hydroxylate lipids and is the first example whose activity increases at higher temperatures (30 degrees C versus 15 degrees C). Thus, while maintaining the hallmark activity of the fatty acyl Delta(12)-desaturase family, the basidiomycete fad2 genes appear to have evolved substantially from an ancestral desaturase.
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Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Phanerochaete/enzimología , Clonación Molecular , Secuencia Conservada , ADN de Hongos/química , ADN de Hongos/genética , Hidroxilación , Intrones , Ácido Linoleico/metabolismo , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Phanerochaete/genética , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
Design of environmentally friendly lubricants derived from renewable resources is highly desirable for many practical applications. Here, Orychophragmus violaceus (Ov) seed oil is found to have superior lubrication properties, and this is based on the unusual structural features of the major lipid species-triacylglycerol (TAG) estolides. Ov TAG estolides contain two non-hydroxylated, glycerol-bound fatty acids (FAs) and one dihydroxylated FA with an estolide branch. Estolide branch chains vary in composition and length, leading to their thermal stability and functional properties. Using this concept, nature-guided estolides of castor oil were synthesized. As predicted, they showed improved lubrication properties similar to Ov seed oil. Our results demonstrate a structure-based design of novel lubricants inspired by natural materials.
RESUMEN
To circumvent the law by evading regulation and obscuring their identities in routine analyses, numerous substituted cathinones have entered the illicit drug market. These compounds have been coined "bath salts" by users. In the described case, the laboratory received an unknown white powder for controlled substances identification. The sample could not be immediately identified using standard methods and procedures. Ultimately, the structure was elucidated using GC-MS, NMR, FTIR, GC-SPIR, UV, and color tests to be 1-(2,3-dihydro-1H-inden-5-yl)-2-(ethylamino)pentan-1-one (bk-IVP), a cathinone analog with a rarely observed nonoxygenated bicyclic ring system. Features of spectra and chemical tests are reported that distinguish this class of cathinones from heterocyclic analogs.
RESUMEN
The biosynthesis of 'unusual' fatty acids with structures that deviate from the common C16 and C18 fatty acids has evolved numerous times in the plant kingdom. Characterization of unusual fatty acid biosynthesis has enabled increased understanding of enzyme substrate properties, metabolic plasticity and oil functionality. Here, we report the identification of a novel pathway for hydroxy fatty acid biosynthesis based on the serendipitous discovery of two C24 fatty acids containing hydroxyl groups at the 7 and 18 carbon atoms as major components of the seed oil of Orychophragmus violaceus, a China-native Brassicaceae. Biochemical and genetic evidence are presented for premature or 'discontinuous' elongation of a 3-OH intermediate by a divergent 3-ketoacyl-CoA (coenzyme A) synthase during a chain extension cycle as the origin of the 7-OH group of the dihydroxy fatty acids. Tribology studies revealed superior high-temperature lubricant properties for O. violaceus seed oil compared to castor oil, a high-performance vegetable oil lubricant. These findings provide a direct pathway for designing a new class of environmentally friendly lubricants and unveil the potential of O. violaceus as a new industrial oilseed crop.
Asunto(s)
Ácidos Grasos/metabolismo , Aceites de Plantas/metabolismo , Semillas/metabolismo , Brassicaceae/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Perfilación de la Expresión Génica , Hidroxilación , Redes y Vías MetabólicasRESUMEN
OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.
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Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Imidazoles/uso terapéutico , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Temozolomida/uso terapéutico , Animales , Neoplasias Encefálicas/patología , Terapia Combinada , Modelos Animales de Enfermedad , Glioblastoma/patología , Humanos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tannins are polyphenols commonly found in plant-derived foods. When ingested they can have various harmful effects, but salivary proline-rich proteins (PRPs) may provide protection against dietary tannins. The aim of this study was to investigate whether basic PRPs, a major family of salivary proteins, can prevent intestinal absorption of tannin. To do so it was necessary first to characterize transport of pentagalloyl glucose (5GG), a hydrolysable tannin, across cultured epithelial cells. Using human intestinal epithelial cells (Caco-2 cells) it was found that a partial degradation of 5GG occurred during transepithelial transport resulting in the presence of 5GG as well as tetra- and trigalloyl glucose and glucose in the receiving compartment. The sodium-dependent glucose transporter SGLT1 played a role in apical (mucosal) to basolateral (serosal) transport and transport in the opposite direction was dependent on the multidrug resistance-associated protein MRP2. An increased uptake from the apical compartment was seen when the basolateral receiving solution was human serum rather than a balanced salt solution. Transport both in apical-basolateral and basolateral-apical directions was reduced when 1B4, a human basic PRP, was added to the 5GG-containing medium. This decrease closely paralleled the formation of insoluble 5GG-1B4 complexes. It appears that the formation of insoluble tannin-protein complexes diminishes the uptake of 5GG and its metabolites. There is little evidence of other biological activities of basic PRPs so in contrast to other salivary proteins they may exert a biological function in the intestines.
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Taninos Hidrolizables/farmacocinética , Absorción Intestinal/efectos de los fármacos , Péptidos/farmacología , Proteínas y Péptidos Salivales/farmacología , Apolipoproteína A-I/metabolismo , Transporte Biológico/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Humanos , Florizina/farmacología , Dominios Proteicos Ricos en Prolina , Unión Proteica , Verapamilo/farmacologíaRESUMEN
Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.
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Imidazoles/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Piperazinas/administración & dosificación , Proteínas Proto-Oncogénicas c-mdm2/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Animales , Carboplatino/administración & dosificación , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Ensayos Clínicos como Asunto , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Histonas/biosíntesis , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Células MCF-7 , Ratones , Metástasis de la Neoplasia , Proteínas Nucleares/genética , Neoplasias de la Mama Triple Negativas/patología , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.
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Materiales Biomiméticos/química , Colesterol/química , Ácidos Grasos Insaturados/química , Membrana Dobles de Lípidos/química , Liposomas/química , Fluidez de la Membrana , Fosfatidilcolinas/química , Deuterio , Campos Electromagnéticos , Membrana Dobles de Lípidos/efectos de la radiación , Liposomas/efectos de la radiación , Espectroscopía de Resonancia Magnética/métodos , Micelas , Conformación Molecular , Transición de Fase , TemperaturaRESUMEN
Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The DeltaFggcs1-null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The DeltaFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild-type PH-1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1-mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host-dependent.
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Defensinas/farmacología , Fusarium/efectos de los fármacos , Glucosilceramidas/metabolismo , Glucosiltransferasas/metabolismo , Medicago sativa/metabolismo , Defensinas/genética , Defensinas/metabolismo , Fusarium/patogenicidad , Prueba de Complementación Genética , Glucosiltransferasas/genética , Solanum lycopersicum/microbiología , Espectroscopía de Resonancia Magnética , Mutación , Triticum/microbiología , Virulencia , Zea mays/microbiologíaRESUMEN
This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal delta12-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with 2H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50), and their dynamic properties were studied in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol % peptide to lipid, 31P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the L(alpha) phase. The 2H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz, indicating increased side-chain motion and solvent exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the 2H-labeled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8 degrees with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, describes the conformation of TM-A in a biomembrane mimic, presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases, and presents a model of arginine and tyrosine control of transmembrane protein stability and insertion.
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Arginina/química , Ácido Graso Desaturasas/química , Membrana Dobles de Lípidos/química , Modelos Moleculares , Tirosina/química , Secuencia de Aminoácidos , Anisotropía , Dimiristoilfosfatidilcolina/química , Microsomas/enzimología , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Fosfolípidos/químicaRESUMEN
The fungal elicitor-induced ELI12 gene from parsley has been previously shown to encode a divergent form of the Delta12-oleic acid desaturase. In this report, we show that the ELI12 gene product is a fatty acid acetylenase or a triple-bond-forming enzyme. Expression of this enzyme in transgenic soybean seeds was accompanied by the accumulation of the Delta12-acetylenic fatty acids, crepenynic and dehydrocrepenynic acids. Using PCR with degenerate oligonucleotides, we also show that homologs of the ELI12 gene are present in other members of the Apiaceae family. In addition, cDNAs for divergent forms of the Delta12-oleic acid desaturase were detected among the expressed sequence tags (ESTs) from English ivy, an Araliaceae species, and sunflower, an Asteraceae species. As with the ELI12 gene, expression of these cDNAs in transgenic soybean embryos was accompanied by the accumulation of crepenynic and dehydrocrepenynic acids. Homologs of the sunflower acetylenase gene were also detected in other Asteraceae species, as revealed by PCR analysis of isolated genomic DNA. Results from Northern blot and EST analyses indicated that the expression of the sunflower gene, like ELI12, was induced by fungal elicitation. Overall, these results demonstrate that expressed genes for Delta12-fatty acid acetylenases occur in at least three plant families, and are responsive to fungal pathogenesis. Natural products derived from crepenynic and dehydrocrepenynic acids that display antifungal, insecticidal, and nematicidal properties are distributed through at least 15 plant families. The acetylenases described here provide probes for chemotaxonomists, and facilitate functional genomic and molecular investigations of these defensive mechanisms.