RESUMEN
The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 µm at 25 °C to 50 µm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.
Asunto(s)
Virus del Dengue/química , Multimerización de Proteína , Proteínas del Envoltorio Viral/química , Virus Zika/química , Temperatura Corporal , Dengue/virología , Humanos , Estabilidad Proteica , Proteínas Recombinantes/química , Vacunas de Subunidad/química , Vacunas Virales/química , Infección por el Virus Zika/virologíaRESUMEN
Controlling which particular members of a large protein family are targeted by a drug is key to achieving a desired therapeutic response. In this study, we report a rational data-driven strategy for achieving restricted polypharmacology in the design of antitumor agents selectively targeting the TYRO3, AXL, and MERTK (TAM) family tyrosine kinases. Our computational approach, based on the concept of fragments in structural environments (FRASE), distills relevant chemical information from structural and chemogenomic databases to assemble a three-dimensional inhibitor structure directly in the protein pocket. Target engagement by the inhibitors designed led to disruption of oncogenic phenotypes as demonstrated in enzymatic assays and in a panel of cancer cell lines, including acute lymphoblastic and myeloid leukemia (ALL/AML) and nonsmall cell lung cancer (NSCLC). Structural rationale underlying the approach was corroborated by X-ray crystallography. The lead compound demonstrated potent target inhibition in a pharmacodynamic study in leukemic mice.
Asunto(s)
Antineoplásicos/química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Estructura Molecular , Neoplasias ExperimentalesRESUMEN
The dengue virus (DENV) causes over 350 million infections, resulting in â¼25,000 deaths per year globally. An effective dengue vaccine requires generation of strong and balanced neutralizing antibodies against all four antigenically distinct serotypes of DENV. The leading live-attenuated tetravalent dengue virus vaccine platform has shown partial efficacy, with an unbalanced response across the four serotypes in clinical trials. DENV subunit vaccine platforms are being developed because they provide a strong safety profile and are expected to avoid the unbalanced immunization issues associated with live multivalent vaccines. Subunit vaccines often lack immunogenicity, requiring either a particulate or adjuvanted formulation. Particulate formulations adsorbing monomeric DENV-E antigen to the particle surface incite a strong immune response, but have no control of antigen presentation. Highly neutralizing epitopes are displayed by DENV-E quaternary structures. To control the display of DENV-E and produce quaternary structures, particulate formulations that covalently attach DENV-E to the particle surface are needed. Here we develop a surface attached DENV2-E particulate formulation, as well as analysis tools, using PEG hydrogel nanoparticles created with particle replication in nonwetting templates (PRINT) technology. We found that adding Tween-20 to the conjugation buffer controls DENV-E adsorption to the particle surface during conjugation, improving both protein stability and epitope display. Immunizations with the anionic but not the cationic DENV2-E conjugated particles were able to produce DENV-specific and virus neutralizing antibody in mice. This work optimized the display of DENV-E conjugated to the surface of a nanoparticle through EDC/NHS chemistry, establishing a platform that can be expanded upon in future work to fully control the display of DENV-E.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Proteínas Inmovilizadas/inmunología , Nanopartículas , Proteínas del Envoltorio Viral/inmunología , Adsorción , Animales , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Chlorocebus aethiops , Dengue/inmunología , Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/química , Virus del Dengue/química , Femenino , Proteínas Inmovilizadas/administración & dosificación , Proteínas Inmovilizadas/química , Inmunización , Ratones Endogámicos BALB C , Modelos Moleculares , Nanopartículas/química , Células Vero , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/químicaRESUMEN
STUDY HYPOTHESIS: Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. STUDY FINDING: This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. WHAT IS KNOWN ALREADY: Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. MAIN RESULTS AND THE ROLE OF CHANCE: Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 µM compared with an IC50 of 38.5 µM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 µM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions). LIMITATIONS, REASONS FOR CAUTION: The chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development. WIDER IMPLICATIONS OF THE FINDINGS: This study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets. LARGE SCALE DATA: None. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Adenosina Trifosfato/metabolismo , Animales , Cristalografía por Rayos X , Glucólisis/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Ratones , Motilidad Espermática/efectos de los fármacosRESUMEN
Computational design of novel protein-protein interfaces is a test of our understanding of protein interactions and has the potential to allow modification of cellular physiology. Methods for designing high-affinity interactions that adopt a predetermined binding mode have proved elusive, suggesting the need for new strategies that simplify the design process. A solvent-exposed backbone on a ß-strand is thought of as "sticky" and ß-strand pairing stabilizes many naturally occurring protein complexes. Here, we computationally redesign a monomeric protein to form a symmetric homodimer by pairing exposed ß-strands to form an intermolecular ß-sheet. A crystal structure of the designed complex closely matches the computational model (rmsd = 1.0 Å). This work demonstrates that ß-strand pairing can be used to computationally design new interactions with high accuracy.
Asunto(s)
Proteínas/química , Cristalografía por Rayos X/métodos , ADN/química , Dimerización , Escherichia coli/metabolismo , Luz , Conformación Molecular , Conformación Proteica , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína , Dispersión de Radiación , Programas Informáticos , Solventes , Propiedades de Superficie , Termodinámica , UltracentrifugaciónRESUMEN
TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to single-stranded DNA (ssDNA) with a site size of â¼25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1-858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Factor F , Adenosina Difosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Sitios de Unión , Contraindicaciones , ADN/metabolismo , ADN Helicasas/genética , ADN de Cadena Simple/metabolismo , Proteínas de Escherichia coli/genética , Polarización de Fluorescencia , Unión Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein α subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/Gα(i) complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted Gα(i1) point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the Gα mutants and GoLoco motifs. For Gα(i1)(Q147L), affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Gα(i1)(Q147L) bound to the RGS14 GoLoco motif revealed disorder among three peptide residues surrounding a well defined Leu-147 side chain. Monte Carlo simulations of the peptide in this region showed a sampling of multiple backbone conformations in contrast to the wild-type complex. We conclude that mutation of Glu-147 to leucine creates a hydrophobic surface favorably buried upon GoLoco peptide binding, yet the hydrophobic Leu-147 also promotes flexibility among residues 511-513 of the RGS14 GoLoco peptide.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Simulación de Dinámica Molecular , Péptidos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Simulación por Computador , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/genética , Humanos , Péptidos/síntesis química , Unión Proteica/genética , Conformación Proteica , TermodinámicaRESUMEN
Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 µM. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (Cα rmsd = 1.4 Å).
Asunto(s)
Diseño de Fármacos , Multimerización de Proteína , Proteínas/química , Zinc , Modelos Moleculares , Estructura Cuaternaria de Proteína , Proteínas/metabolismoRESUMEN
Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The DeltahacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37 degrees C, but cell wall integrity was disrupted at 45 degrees C, resulting in a dramatic loss in viability. The DeltahacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the DeltahacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.
Asunto(s)
Aspergillus fumigatus/patogenicidad , Retículo Endoplásmico/fisiología , Pliegue de Proteína , Animales , Aspergilosis/etiología , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/microbiología , Homeostasis , Ratones , VirulenciaRESUMEN
Major histocompatibility complex (MHC) class I variants H-2K(b) and H-2K(bm8) differ primarily in the B pocket of the peptide-binding groove, which serves to sequester the P2 secondary anchor residue. This polymorphism determines resistance to lethal herpes simplex virus (HSV-1) infection by modulating T cell responses to the immunodominant glycoprotein B(498-505) epitope, HSV8. We studied the molecular basis of these effects and confirmed that T cell receptors raised against K(b)-HSV8 cannot recognize H-2K(bm8)-HSV8. However, substitution of Ser(P2) to Glu(P2) (peptide H2E) reversed T cell receptor (TCR) recognition; H-2K(bm8)-H2E was recognized whereas H-2K(b)-H2E was not. Insight into the structural basis of this discrimination was obtained by determining the crystal structures of all four MHC class I molecules in complex with bound peptide (pMHCs). Surprisingly, we find no concerted pMHC surface differences that can explain the differential TCR recognition. However, a correlation is apparent between the recognition data and the underlying peptide-binding groove chemistry of the B pocket, revealing that secondary anchor residues can profoundly affect TCR engagement through mechanisms distinct from the alteration of the resting state conformation of the pMHC surface.
Asunto(s)
Antígenos H-2/química , Receptores de Antígenos de Linfocitos T/química , Proteínas del Envoltorio Viral/inmunología , Alelos , Animales , Dicroismo Circular , Cristalografía por Rayos X , Enlace de Hidrógeno , Epítopos Inmunodominantes , Conformación Proteica , Linfocitos T/inmunologíaRESUMEN
Human UDP-glucuronosyltransferases (EC 2.4.1.17) (UGTs) are major phase II metabolism enzymes that detoxify a multitude of endo- and xenobiotics through the covalent addition of a glucuronic acid moiety. UGTs are promiscuous enzymes that regulate the levels of numerous important endobiotics in a range of tissues, and inactivate most therapeutic compounds in concert with phase I enzymes. In spite of the importance of these enzymes, we have only a limited understanding of the molecular mechanisms governing their substrate specificity and catalytic activity. Until recently, no three-dimensional structural information was available for any mammalian UGT. The 1.8-å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT2B7 (2B7CT) is the only structure of a mammalian UGT target determined to date. In this review, we summarize what has been learned about human UGT function from the analysis of this and other related glycosyltransferase (GT) crystal structures.
Asunto(s)
Glucuronosiltransferasa/metabolismo , Animales , Sitios de Unión/genética , Cristalografía por Rayos X , Glucuronosiltransferasa/química , Humanos , Mamíferos , Modelos Moleculares , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Xenobióticos/metabolismoRESUMEN
The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies, most likely because the antigens tested do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. Here, we produce stable recombinant E protein homodimers that are recognized by strongly neutralizing Zika specific monoclonal antibodies. In mice, the dimeric antigen stimulate strongly neutralizing antibodies that target epitopes that are similar to epitopes recognized by human antibodies following natural Zika virus infection. The monomer antigen stimulates low levels of E-domain III targeting neutralizing antibodies. In a Zika challenge model, only E dimer antigen stimulates protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines.
Asunto(s)
Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Chlorocebus aethiops , Epítopos/inmunología , Femenino , Humanos , Ratones Endogámicos C57BL , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Células Vero , Proteínas del Envoltorio Viral/genética , Virus Zika/genética , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in processing a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8-A resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designated UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicates human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.
Asunto(s)
Glucuronosiltransferasa/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Análisis Mutacional de ADN , Glucuronatos/química , Glucuronatos/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Uridina Difosfato/química , Uridina Difosfato/metabolismoRESUMEN
All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the (393)DQMDNAK(399) region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q(394),D(396), and K(399) on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D(393) (D(398) in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q(394) (Q(399) in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D(393) is critical for the binding of glucuronic acid and that proximal residues, e.g., Q(394) (Q(399) in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism.
Asunto(s)
Ácido Aspártico/metabolismo , Glucuronosiltransferasa/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Catálisis , Clonación Molecular , Glucuronosiltransferasa/química , Glucuronosiltransferasa/genética , Humanos , Isoenzimas , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Uridina Difosfato Ácido Glucurónico/químicaRESUMEN
Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The DeltaAfatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the DeltaAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the DeltaAfatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the DeltaAfatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.
Asunto(s)
Antígenos Fúngicos/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Autofagia , Iones/química , Metales/química , Proteínas Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Animales , Proteínas Relacionadas con la Autofagia , Cationes , Ácido Edético/química , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Nitrógeno/química , Oligonucleótidos/química , Proteínas Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidad de la EspecieRESUMEN
P-glycoprotein (Pgp) has been considered as a major cause of cancer multidrug resistance; however, clinical solutions to overcome this drug resistance do not exist despite the tremendous endeavors. The lack of cancer specificity is a main reason for clinical failure of conventional approaches. Targeted photodynamic therapy (PDT) is highly cancer specific by combining antibody targeting and locoregional light irradiation. We aimed to develop Pgp-targeted PDT using antibody-photosensitizer conjugates made of a recombinant Fab fragment. We prepared the photosensitizer conjugates by expressing a recombinant Fab fragment and specifically linking IR700-maleimide at the C-terminal of the Fab heavy chain. In vitro studies showed that the Fab conjugates specifically bind to Pgp. Their phototoxicity was comparable to full antibody conjugates when assayed with conventional 2-D cell culture, but they outperformed the full antibody conjugates in a 3-D tumor spheroid model. In a mouse xenograft model of chemoresistant tumors, Fab conjugates showed Pgp specific delivery to chemoresistant tumors. Upon irradiation with near-infrared light, they caused rapid tumor shrinkage and significantly prolonged the survival of tumor-bearing mice. Compared to the full antibody conjugates, Fab conjugates took a shorter time to reach peak tumor levels and achieved a more homogeneous tumor distribution. This allows light irradiation to be initiated at a shorter time interval after the conjugate injection, and thus may facilitate clinical translation. We conclude that our targeted PDT approach provides a highly cancer-specific approach to combat chemoresistant tumors, and that the conjugates made of recombinant antibody fragments are superior to full antibody conjugates for targeted PDT.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Anticuerpos/química , Antineoplásicos/química , Resistencia a Antineoplásicos , Fragmentos Fab de Inmunoglobulinas/química , Fotoquimioterapia/métodos , Células 3T3 , Animales , Anticuerpos/uso terapéutico , Antineoplásicos/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Terapia Combinada/métodos , Resistencia a Múltiples Medicamentos , Femenino , Xenoinjertos , Humanos , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/diagnóstico por imagen , Neoplasias/patología , Neoplasias/terapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéutico , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Distribución TisularRESUMEN
Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic shock syndrome. Dengue vaccine development is challenging because of the need to induce protection against four antigenically distinct DENV serotypes. Recent studies indicate that tetravalent DENV vaccines must induce balanced, serotype-specific neutralizing antibodies to achieve durable protective immunity against all 4 serotypes. With the leading live attenuated tetravalent DENV vaccines, it has been difficult to achieve balanced and type-specific responses to each serotype, most likely because of unbalanced replication of vaccine viral strains. Here we evaluate a tetravalent DENV protein subunit vaccine, based on recombinant envelope protein (rE) adsorbed to the surface of poly (lactic-co-glycolic acid) (PLGA) nanoparticles for immunogenicity in mice. In monovalent and tetravalent formulations, we show that particulate rE induced higher neutralizing antibody titers compared to the soluble rE antigen alone. Importantly, we show the trend that tetravalent rE adsorbed to nanoparticles stimulated a more balanced serotype specific antibody response to each DENV serotype compared to soluble antigens. Our results demonstrate that tetravalent DENV subunit vaccines displayed on nanoparticles have the potential to overcome unbalanced immunity observed for leading live-attenuated vaccine candidates.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Nanopartículas/administración & dosificación , Proteínas Estructurales Virales/inmunología , Animales , Vacunas contra el Dengue/administración & dosificación , Femenino , Ratones Endogámicos BALB C , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/administración & dosificación , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Dengue viruses (DENVs) are mosquito-borne flaviviruses and the causative agents of dengue fever and dengue hemorrhagic fever. As there are four serotypes of DENV (DENV1-4), people can be infected multiple times, each time with a new serotype. Primary infections stimulate antibodies that mainly neutralize the serotype of infection (type-specific), whereas secondary infections stimulate responses that cross-neutralize 2 or more serotypes. Previous studies have demonstrated that neutralizing antibodies induced by primary infections recognize tertiary and quaternary structure epitopes on the viral envelope (E) protein that are unique to each serotype. The goal of the current study was to determine the properties of neutralizing antibodies induced after secondary infection with a different (heterotypic) DENV serotypes. We evaluated whether polyclonal neutralizing antibody responses after secondary infections consist of distinct populations of type-specific antibodies to each serotype encountered or a new population of broadly cross-neutralizing antibodies. We observed two types of responses: in some individuals exposed to secondary infections, DENV neutralization was dominated by cross-reactive antibodies, whereas in other individuals both type-specific and cross-reactive antibodies contributed to neutralization. To better understand the origins of type-specific and cross-reactive neutralizing antibodies, we analyzed sera from individuals with well-documented sequential infections with two DENV serotypes only. These individuals had both type-specific and cross-reactive neutralizing antibodies to the 2 serotypes responsible for infection and only cross-reactive neutralizing antibodies to other serotypes. Collectively, the results demonstrate that the quality of neutralizing (and presumably protective) antibodies are different in individuals depending on the number of previous exposures to different DENV serotypes. We propose a model in which low affinity, cross-reactive antibody secreting B-cell clones induced by primary exposure evolve during each secondary infection to secrete higher affinity and more broadly neutralizing antibodies.
Asunto(s)
Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Coinfección/sangre , Virus del Dengue/clasificación , Dengue/inmunología , Adolescente , Anticuerpos Neutralizantes/sangre , Niño , Preescolar , Estudios de Cohortes , Reacciones Cruzadas , Dengue/epidemiología , Dengue/virología , Humanos , Lactante , Serogrupo , ViajeRESUMEN
Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development.
Asunto(s)
Virus del Dengue , Multimerización de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus , Virus Zika , Animales , Anticuerpos Antivirales/inmunología , Sitios de Unión , Células Cultivadas , Virus del Dengue/clasificación , Virus del Dengue/fisiología , Epítopos/inmunología , Humanos , Pruebas de Neutralización , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteínas Recombinantes , Serogrupo , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/inmunología , Virus Zika/clasificación , Virus Zika/fisiologíaRESUMEN
Macrocycles have attracted significant attention in drug discovery recently. In fact, a few deâ novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure-based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)-specific inhibitors. An enzyme-linked immunosorbent assay (ELISA) in 384-well format was employed to evaluate the inhibitory activity of macrocycles in a cell-based assay assessing tyrosine phosphorylation of MerTK. Through structure-activity relationship (SAR) studies, analogue 11 [UNC2541; (S)-7-amino-N-(4-fluorobenzyl)-8-oxo-2,9,16-triaza-1(2,4)-pyrimidinacyclohexadecaphane-1-carboxamide] was identified as a potent and MerTK-specific inhibitor that exhibits sub-micromolar inhibitory activity in the cell-based ELISA. In addition, an X-ray structure of MerTK protein in complex with 11 was resolved to show that these macrocycles bind in the MerTK ATP pocket.