RESUMEN
Cancer-associated alterations to glycosylation have been shown to aid cancer development and progression. An increased abundance of high mannose N-glycans has been observed in several cancers. Here, we describe the preparation of lectin drug conjugates (LDCs) that permit toxin delivery to cancer cells presenting high mannose N-glycans. Additionally, we demonstrate that cancer cells presenting low levels of high mannose N-glycans can be rendered sensitive to the LDCs by co-treatment with a type I mannosidase inhibitor. Our findings establish that an increased abundance of high mannose N-glycans in the glycocalyx of cancer cells can be leveraged to enable toxin delivery.
Asunto(s)
Lectinas , Manosa , Manosidasas , Preparaciones Farmacéuticas , PolisacáridosRESUMEN
An engineered cyanovirin-N homologue that exhibits specificity for high mannose N-glycans has been constructed to aid typeâ I α 1,2-mannosidase inhibitor discovery and development. Engineering the lectins C-terminus permitted facile functionalization with fluorophores via a sortase and click strategy. The resulting lectin constructs exhibit specificity for cells presenting high mannose N-glycans. Importantly, these lectin constructs can also be applied to specifically assess changes in cell surface glycosylation induced by typeâ I mannosidase inhibitors. Testing the utility of these lectin constructs led to the discovery of typeâ I mannosidase inhibitors with nanomolar potency. Cumulatively, these findings reveal the specificity and utility of the functionalized cyanovirin-N homologue constructs, and highlight their potential in analytical contexts that require high mannose-specific lectins.
Asunto(s)
Lectinas/química , Manosidasas/antagonistas & inhibidores , Alcaloides/química , Alcaloides/metabolismo , Secuencias de Aminoácidos , Aminoaciltransferasas/química , Proteínas Bacterianas/química , Línea Celular , Cisteína Endopeptidasas/química , Diseño de Fármacos , Colorantes Fluorescentes/química , Glicosilación , Humanos , Lectinas/metabolismo , Manosa/química , Manosa/metabolismo , Manosidasas/metabolismo , Microscopía FluorescenteRESUMEN
The RNA-binding protein Hu antigen R (HuR) is a post-transcriptional regulator critical in several types of diseases, including cancer, making it a promising therapeutic target. We have identified small-molecule inhibitors of HuR through a screening approach used in combination with fragment analysis. A total of 36 new compounds originating from fragment linking or structural optimization were studied to establish structure-activity relationships in the set. Two top inhibitors, 1c and 7c, were further validated by binding assays and cellular functional assays. Both block HuR function by directly binding to the RNA-binding pocket, inhibit cancer cell growth dependence of HuR, and suppress cancer cell invasion. Intraperitoneal administration of inhibitor 1c inhibits tumor growth as a single agent and shows a synergistic effect in combination with chemotherapy docetaxel in breast cancer xenograft models. Mechanistically, 1c interferes with the HuR-TGFB/THBS1 axis.
Asunto(s)
Neoplasias , Humanos , Xenoinjertos , Transformación Celular Neoplásica , Línea Celular TumoralRESUMEN
The brain during Alzheimer's disease (AD) is under severe oxidative attack by reactive oxygen species that may lead to methionine oxidation. Oxidation of the sole methionine (Met35) of beta-amyloid (Aß), and possibly methionine residues of other extracellular proteins, may be one of the earliest events contributing to the toxicity of Aß and other proteins in vivo. In the current study, we immunized transgenic AD (APP/PS1) mice at 4 months of age with a recombinant methionine sulfoxide (MetO)-rich protein from Zea mays (antigen). This treatment induced the production of anti-MetO antibody in blood-plasma that exhibits a significant titer up to at least 10 months of age. Compared to the control mice, the antigen-injected mice exhibited the following significant phenotypes at 10 months of age: better short and long memory capabilities; reduced Aß levels in both blood-plasma and brain; reduced Aß burden and MetO accumulations in astrocytes in hippocampal and cortical regions; reduced levels of activated microglia; and elevated antioxidant capabilities (through enhanced nuclear localization of the transcription factor Nrf2) in the same brain regions. These data collected in a preclinical AD model are likely translational, showing that active immunization could give a possibility of delaying or preventing AD onset. This study represents a first step toward the complex way of starting clinical trials in humans and conducting the further confirmations that are needed to go in this direction.
RESUMEN
Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.
Asunto(s)
Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/aislamiento & purificación , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Cromatografía de Afinidad , Clonación Molecular , Escherichia coli/genética , Regulación hacia ArribaRESUMEN
Due to their high hydrophobicity, it is a challenge to obtain high yields of transmembrane peptides for structural and functional characterization. In the present work, a robust method is developed for the expression, purification and reconstitution of transmembrane peptides, especially for those containing conserved methionines. By using a truncated and mutated glutathione-S-transferase construct as the carrier protein and hydroxylamine (which specifically cleaves the peptide bond between Asn and Gly) as the cleavage reagent, 10 mg of the first transmembrane helix of CorA, a Mg2+ transporter from Mycobacterium tuberculosis, can be conveniently obtained with high purity from 1 L of M9 minimal media under optimized conditions. The biophysical properties of the peptide were studied by circular dichroism and nuclear magnetic resonance spectroscopy, and the results show that this CorA peptide is well folded in detergent micelles and the secondary structure is very similar to that in recent crystal structures. In addition, this CorA construct is oligomeric in perfluoro-octanoic acid micelles. The compatibility with the transmembrane peptides containing conserved methionines, the high yield and the simple process make the present method competitive with other commonly used methods to produce such peptides for structural and functional studies.
Asunto(s)
Secuencia Conservada , Hidroxilamina/metabolismo , Metionina/metabolismo , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Caprilatos , Fluorocarburos , Glutatión Transferasa/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/químicaRESUMEN
Membrane protein function within the membrane interstices is achieved by mechanisms that are not typically available to water-soluble proteins. The whole balance of molecular interactions that stabilize three-dimensional structure in the membrane environment is different from that in an aqueous environment. As a result interhelical interactions are often dominated by non-specific van der Waals interactions permitting dynamics and conformational heterogeneity in these interfaces. Here, solid-state NMR data of the transmembrane domain of the M2 protein from influenza A virus are used to exemplify such conformational plasticity in a tetrameric helical bundle. Such data lead to very high resolution structural restraints that can identify both subtle and substantial structural differences associated with various states of the protein. Spectra from samples using two different preparation protocols, samples prepared in the presence and absence of amantadine, and spectra as a function of pH are used to illustrate conformational plasticity.
Asunto(s)
Canales Iónicos/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Matriz Viral/química , Virus de la Influenza A/metabolismo , Modelos Teóricos , Isótopos de Nitrógeno , Estructura Terciaria de ProteínaRESUMEN
Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.
Asunto(s)
Proteínas de la Membrana/química , Péptidos/química , Secuencia de Aminoácidos , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Micelas , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Membrane protein structure and function is critically dependent on the surrounding environment. Consequently, utilizing a membrane mimetic that adequately models the native membrane environment is essential. A range of membrane mimetics are available but none generates a better model of native aqueous, interfacial, and hydrocarbon core environments than synthetic lipid bilayers. Transmembrane α-helices are very stable in lipid bilayers because of the low water content and low dielectric environment within the bilayer hydrocarbon core that strengthens intrahelical hydrogen bonds and hinders structural rearrangements within the transmembrane helices. Recent evidence from solid-state NMR spectroscopy illustrates that transmembrane α-helices, both in peptides and full-length proteins, appear to be highly uniform based on the observation of resonance patterns in PISEMA spectra. Here, we quantitate for the first time through simulations what we mean by highly uniform structures. Indeed, helices in transmembrane peptides appear to have backbone torsion angles that are uniform within ± 4°. While individual helices can be structurally stable due to intrahelical hydrogen bonds, interhelical interactions within helical bundles can be weak and nonspecific, resulting in multiple packing arrangements. Some helical bundles have the capacity through their amino acid composition for hydrogen bonding and electrostatic interactions to stabilize the interhelical conformations and solid-state NMR data is shown here for both of these situations. Solid-state NMR spectroscopy is unique among the techniques capable of determining three-dimensional structures of proteins in that it provides the ability to characterize structurally the membrane proteins at very high resolution in liquid crystalline lipid bilayers.
Asunto(s)
Materiales Biomiméticos/química , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Péptidos/química , Fosfolípidos/química , Secuencia de Aminoácidos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
Sample instability during solid-state NMR experiments frequently arises due to RF heating in aligned samples of hydrated lipid bilayers. A new, simple approach for estimating sample temperature is used to show that, at 9.4 T, sample heating depends mostly on (1)H decoupling power rather than on (15)N irradiation in PISEMA experiments. Such heating for different sample preparations, including lipid composition, salt concentration and hydration level was assessed and the hydration level was found to be the primary parameter correlated with sample heating. The contribution to RF heating from the dielectric loss appears to be dominant under our experimental conditions. The heat generated by a single scan was approximately calculated from the Q values of the probe, to be a 1.7 degrees C elevation per single pulse sequence iteration under typical sample conditions. The steady-state sample temperature during PISEMA experiments can be estimated based on the method presented here, which correlates the loss factor with the temperature rise induced by the RF heating of the sample.
Asunto(s)
Membrana Dobles de Lípidos/química , Resonancia Magnética Nuclear Biomolecular/métodos , Calor , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Ondas de Radio , Cloruro de Sodio , TemperaturaRESUMEN
CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon-nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. These reactions all involve attack on a cyano or carbonyl carbon by a conserved novel catalytic triad Glu-Lys-Cys through a thiol acylenzyme intermediate. However, classification into the CN-hydrolase superfamily based on sequence similarity alone is not straightforward and further structural data are necessary to improve this categorization. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC1258, a CN-hydrolase superfamily protein from the plant pathogen Xanthomonas campestris (Xcc), are reported. The SeMet-substituted XC1258 crystals diffracted to a resolution of 1.73 A. They are orthorhombic and belong to space group P2(1)2(1)2, with unit-cell parameters a = 143.8, b = 154.63, c = 51.3 A, respectively.
Asunto(s)
Proteínas Bacterianas/química , Hidrolasas/química , Xanthomonas campestris/enzimología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Caenorhabditis elegans/genética , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Hidrolasas/genética , Hidrolasas/aislamiento & purificación , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Xanthomonas campestris is a Gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. It is therefore important to identify potential pathogenic factors involved in this plant disease. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2113, a YaeQ protein possibly involved in the production of virulence factors in Xanthomonas campestris pathovar campestris, are reported. The XC2113 crystals diffracted well to a resolution of at least 1.28 A. They are orthorhombic and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.86, b = 62.69, c = 79.96 A.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Xanthomonas campestris/química , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Cristalización , Cristalografía por Rayos XRESUMEN
Histidine-triad (HIT) proteins are a superfamily of nucleotide hydrolases and transferases that contain a conserved Hphi Hphi Hphi phi motif (where phi is a hydrophobic amino acid) and are found in a variety of organisms. In addition to binding to a variety of nucleotides, other biological functions of the HIT superfamily proteins have been discovered and HIT malfunction has been implicated in several human diseases. Structural studies of HIT superfamily proteins are thus of particular interest. In this manuscript, the cloning, expression, crystallization and preliminary X-ray analysis of XC1015, a HIT protein present in the plant pathogen Xanthomonas campestris pathovar campestris, are reported. The XC1015 crystals diffracted to a resolution of 1.3 A. They are tetragonal and belong to space group P4(3)2(1)2, with unit-cell parameters a = 40.52, b = 40.52, c = 126.89 A.
Asunto(s)
Proteínas Bacterianas/química , Hidrolasas/química , Xanthomonas campestris/química , Cristalización , Cristalografía por Rayos XRESUMEN
Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6 A. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73 A.
Asunto(s)
Proteínas Bacterianas/química , Cobre/metabolismo , Xanthomonas campestris/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Homeostasis , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Programas Informáticos , Difracción de Rayos X/métodosRESUMEN
Xanthomonas campestris pv. campestris is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, roughly one third of which have no known structure and/or function. However, some genes of unknown function are highly conserved among several different bacterial genuses. XC6422 is one such conserved hypothetical protein and has been overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. Crystals grew to approximately 2 x 1.5 x 0.4 mm in size after one week and diffracted to at least 1.6 A resolution. They belong to the monoclinic space group C2, with one molecule per asymmetric unit and unit-cell parameters a = 75.8, b = 79.3, c = 38.2 A, beta = 109.4 degrees. Determination of this structure may provide insights into the protein's function.
Asunto(s)
Cristalografía por Rayos X/métodos , Xanthomonas campestris/metabolismo , Proteínas Bacterianas/química , Clonación Molecular , Cristalización , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Xanthomonas campestris/genéticaRESUMEN
Xanthomonas campestris pv. campestris strain 17 is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, one third of which have no known structure and/or function yet are highly conserved among several different bacterial genuses. One of these gene products is XC1692 protein, containing 141 amino acids. It was overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. The crystals diffract to at least 1.45 A resolution. They are hexagonal and belong to space group P6(3), with unit-cell parameters a = b = 56.9, c = 71.0 A. They contain one molecule per asymmetric unit.
Asunto(s)
Proteínas Bacterianas/química , Xanthomonas campestris/química , Proteínas Bacterianas/genética , Clonación Molecular , Cristalografía por Rayos X , Difusión , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Genoma Bacteriano , Conformación Proteica , Difracción de Rayos XRESUMEN
The multiple antibiotic resistance operon (marRAB) is a member of the multidrug-resistance system. When induced, this operon enhances resistance of bacteria to a variety of medically important antibiotics, causing a serious global health problem. MarR is a marR-encoded protein that represses the transcription of the marRAB operon. Through binding with salicylate and certain antibiotics, however, MarR can derepress and activate the marRAB operon. In this report, the cloning, expression, crystallization and preliminary X-ray analysis of XC1739, a putative MarR repressor protein present in the Xanthomonas campestris pv. campestris, a Gram-negative bacterium causing major worldwide disease of cruciferous crops, are described. The XC1739 crystals diffracted to a resolution of at least 1.8 A. They are orthorhombic and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 39.5, b = 54.2 and c = 139.5 A, respectively. They contain two molecules in the asymmetric unit from calculation of the self-rotation function.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Farmacorresistencia Microbiana , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Xanthomonas campestris/química , Clonación Molecular , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Operón , Conformación Proteica , Proteínas Represoras/química , Difracción de Rayos X , Xanthomonas campestris/genéticaRESUMEN
Oligoribonucleases are essential components of RNA and DNA metabolism and close homologues of genes encoding them are found not only in prokaryotes but also in a wide range of eukaryotes, including yeast and humans. Inactivation of the oligoribonuclease gene (orn) can result in cellular lethality. Despite their important biological function, they have been studied little from a structural point of view. In this report, the cloning, expression, crystallization and preliminary X-ray analysis of XC847, a DEDDh-type 3'-5' oligoribonuclease from the plant pathogen Xanthomonas campestris pv. campestris, a Gram-negative bacterium causing major worldwide disease of cruciferous crops, is described. The XC847 crystals diffracted to a resolution of at least 2.1 A. They are tetragonal and belong to space group P4(3)2(1)2, with unit-cell parameters a = b = 67.5, c = 89.8 A. One molecule is present per asymmetric unit.
Asunto(s)
Exorribonucleasas/química , Xanthomonas campestris/enzimología , Secuencias de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , ADN/química , Electroforesis en Gel de Poliacrilamida , ARN/química , Difracción de Rayos XRESUMEN
CorA is a constitutively expressed magnesium transporter in many bacteria. The crystal structures of Thermotoga maritima CorA provide an excellent structural framework for continuing studies. Here, the ligand binding properties of the conserved interhelical loop, the only portion of the protein exposed to the periplasmic space, are characterized by solution nuclear magnetic resonance spectroscopy. Through titration experiments performed on the isolated transmembrane domain of Mycobacterium tuberculosis CorA, it was found that two CorA substrates (Mg2+ and Co2+) and the CorA-specific inhibitor (Co(III) hexamine chloride) bind in the loop at the same binding site. This site includes the glutamic acid residue from the conserved "MPEL" motif. The relatively large dissociation constants indicate that such interactions are weak but not atypical for channels. The present data support the hypothesis that the negatively charged loop could act as an electrostatic ring, increasing local substrate concentrations before transport across the membrane.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Magnesio/metabolismo , Mycobacterium tuberculosis/metabolismo , Thermotoga maritima/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Dicroismo Circular , Clonación Molecular , Cobalto/metabolismo , Secuencia Conservada , Reactivos de Enlaces Cruzados/farmacología , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mycobacterium tuberculosis/genética , Unión Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.