RESUMEN
Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias de la Próstata Resistentes a la Castración , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Humanos , Ligandos , Masculino , Ratones , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Linfocitos TRESUMEN
MsbA is an essential ATP-binding cassette transporter in Gram-negative bacteria that transports lipid A and lipopolysaccharide from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane. Here we report the X-ray structure of MsbA from Salmonella typhimurium at 2.8-Å resolution in an inward-facing conformation after cocrystallization with lipid A and using a stabilizing facial amphiphile. The structure displays a large amplitude opening in the transmembrane portal, which is likely required for lipid A to pass from its site of synthesis into the protein-enclosed transport pathway. Putative lipid A density is observed further inside the transmembrane cavity, consistent with a trap and flip model. Additional electron density attributed to lipid A is observed near an outer surface cleft at the periplasmic ends of the transmembrane helices. These findings provide new structural insights into the lipid A transport pathway through comparative analysis with existing MsbA structures.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Proteínas Bacterianas/química , Membrana Celular/química , Lípido A/química , Proteínas de Transferencia de Fosfolípidos/química , Salmonella typhimurium/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Transporte Biológico , Membrana Celular/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Lípido A/metabolismo , Modelos Moleculares , Periplasma/química , Periplasma/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the "rest" period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.
Asunto(s)
Bioingeniería/métodos , Inmunoterapia Adoptiva/métodos , Animales , Antígenos CD19/inmunología , Linfocitos B/inmunología , Citocinas/metabolismo , Citotoxicidad Inmunológica/inmunología , Femenino , Región de Cambio de la Inmunoglobulina/genética , Región de Cambio de la Inmunoglobulina/inmunología , Activación de Linfocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Modelos Biológicos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunologíaRESUMEN
Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 °C and 20 °C.
RESUMEN
P-glycoprotein (P-gp) is a transporter of great clinical and pharmacological significance. Several structural studies of P-gp and its homologs have provided insights into its transport cycle, but questions remain regarding how P-gp recognizes diverse substrates and how substrate binding is coupled to ATP hydrolysis. Here, four new P-gp co-crystal structures with a series of rationally designed ligands are presented. It is observed that the binding of certain ligands, including an ATP-hydrolysis stimulator, produces a large conformational change in the fourth transmembrane helix, which is positioned to potentially transmit a signal to the nucleotide-binding domains. A new ligand-binding site on the surface of P-gp facing the inner leaflet of the membrane is also described, providing vital insights regarding the entry mechanism of hydrophobic drugs and lipids into P-gp. These results represent significant advances in the understanding of how P-gp and related transporters bind and export a plethora of metabolites, antibiotics and clinically approved and pipeline drugs.
Asunto(s)
Adenosina Trifosfato/química , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Cristalografía por Rayos X , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/ultraestructura , Proteínas Bacterianas/ultraestructura , Transportadoras de Casetes de Unión a ATP/química , Animales , Proteínas Bacterianas/química , Lípidos de la Membrana/química , Ratones , Microscopía Electrónica , Modelos Moleculares , Nucleótidos/química , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Homología Estructural de ProteínaRESUMEN
We designed ß-strand peptides that stabilize integral membrane proteins (IMPs). ß-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-ß-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. ß-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.
Asunto(s)
Proteínas de la Membrana/química , Nanoestructuras/química , Péptidos/química , Péptidos/síntesis química , Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Dicroismo Circular , Proteínas de la Membrana/síntesis química , Microscopía Electrónica de Transmisión , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroid-based facial amphiphiles (FAs) that are structurally distinct from conventional detergents and previously developed FAs. The unique FAs stabilize MPs and form relatively small protein-detergent complexes (PDCs), a property considered favorable for MP crystallization. We attempted to crystallize several MPs belonging to different protein families, including the human gap junction channel protein connexin 26, the ATP binding cassette transporter MsbA, the seven-transmembrane G protein-coupled receptor-like bacteriorhodopsin, and cytochrome P450s (peripheral MPs). Using FAs alone or mixed with other detergents or lipids, we obtained 3D crystals of the above proteins suitable for X-ray crystallographic analysis. The fact that FAs enhance MP crystallizability compared with traditional detergents can be attributed to several properties, including increased protein stability, formation of small PDCs, decreased PDC surface flexibility, and potential to mediate crystal lattice contacts.
Asunto(s)
Cristalografía por Rayos X/métodos , Uniones Comunicantes/química , Proteínas de la Membrana/química , Esteroides/química , Tensoactivos/química , Humanos , Estabilidad ProteicaRESUMEN
Sugar-based detergents, mostly derived from maltose or glucose, prevail in the extraction, solubilization, stabilization, and crystallization of membrane proteins. Inspired by the broad use of trehalose for protecting biological macromolecules and lipid bilayer structures, we synthesized new trehaloside detergents for potential applications in membrane protein research. We devised an efficient synthesis of four dodecyl trehalosides, each with the 12-carbon alkyl chain attached to different hydroxyl groups of trehalose, thus presenting a structurally diverse but related family of detergents. The detergent physical properties, including solubility, hydrophobicity, critical micelle concentration (CMC), and size of micelles, were evaluated and compared with the most popular maltoside analogue, ß-D-dodecyl maltoside (DDM), which varied from each other due to distinct molecular geometries and possible polar group interactions in resulting micelles. Crystals of 2-dodecyl trehaloside (2-DDTre) were also obtained in methanol, and the crystal packing revealed multiple H-bonded interactions among adjacent trehalose groups. The few trehaloside detergents were tested for the solubilization and stabilization of the nociceptin/orphanin FQ peptide receptor (ORL1) and MsbA, which belong to the G-protein coupled receptor (GPCR) and ATP-binding cassette transporter families, respectively. Our results demonstrated the utility of trehaloside detergents as membrane protein solubilization reagents with the optimal detergents being protein dependent. Continuing development and investigations of trehaloside detergents are attractive, given their interesting and unique chemical-physical properties and potential interactions with membrane lipids.
Asunto(s)
Detergentes/química , Glucósidos/química , Proteínas de la Membrana/química , Receptores Acoplados a Proteínas G/química , Receptores Opioides/química , Receptor de NociceptinaRESUMEN
An instrument to measure dynamic adhesive forces between interacting rough surfaces has been developed. It consists of four parts, namely, main instrument body, vertical positioning system with both micrometer and nanometer positioning accuracies, horizontal positioning system with nanometer positioning accuracy, and custom-built high-resolution, and high dynamic bandwidth capacitive force transducer. The vertical piezoelectric actuator (PZT) controls the vertical (approaching and retracting) motion of the upper specimen, while the horizontal PZT controls the horizontal (reciprocal) motion of the lower specimen. The force transducer is placed in line with the upper specimen and vertical PZT, and directly measures the adhesive forces with a root-mean-square load resolution of 1.7 microN and a dynamic bandwidth of 1.7 kHz. The newly developed instrument enables reliable measurements of near-contact and contact adhesive forces for microscale devices under different dynamic conditions. Using the developed instrument, dynamic pull-in and pull-off force measurements were performed between an aluminum-titanium-carbide sphere and a 10 nm thick carbon film disk sample. Three different levels of contact force were investigated; where for each contact force level the vertical velocity of the upper sample was varied from 0.074 to 5.922 microms, while the lower sample was stationary. It was found that slower approaching and retracting velocities result in higher pull-in and pull-off forces. The noncontact attractive force was also measured during horizontal movement of the lower sample, and it was found that the periodic movements of the lower disk sample also affect the noncontact surface interactions.
RESUMEN
We have previously reported that Fas-resistant A20 cells (FasR) have phospholipase D (PLD) activity upregulated by endogenous PLD2 overexpression. In the present study, we investigated how overexpressed PLD2 in FasR could generate survival signals by regulating the protein levels of anti-apoptotic Bcl-2 and Bcl-xL. To confirm the effect of PLD2 on Bcl-2 protein levels, we transfected PLD2 into wild-type murine B lymphoma A20 cells. The transfected cells showed markedly the increases in Bcl-2 and Bcl-xL protein levels, and became resistant to Fas-induced apoptosis, similar to FasR. Treatment of wild-type A20 cells with phosphatidic acid (PA), the metabolic end product of PLD2 derived from phosphatidylcholin, markedly increased levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Moreover, PA-induced expressions of Bcl-2 and Bcl-xL were enhanced by propranolol, an inhibitor of PA phospholydrolase (PAP), whereas completely blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), suggesting that PLA(2) metabolite of PA is responsible for the increases in Bcl-2 and Bcl-xL protein levels. We further confirmed the involvement of arachidonic acid (AA) in PA-induced survival signals by showing that 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), PA without AA, was unable to increase Bcl-2 and Bcl-xL proteins. Moreover, PA notably increased cyclooxygenase (COX)-2 protein expression, and PA-induced expression of both Bcl-2 and Bcl-xL was inhibited by NS-398, a specific inhibitor of COX-2. Taken together, these findings demonstrate that PA generated by PLD2 plays an important role in cell survival during Fas-mediated apoptosis through the increased Bcl-2 and Bcl-xL protein levels which resulted from PLA(2) and AA-COX2 pathway.
Asunto(s)
Apoptosis/fisiología , Fosfolipasa D/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/fisiología , Proteína bcl-X/metabolismo , Animales , Antihipertensivos/metabolismo , Línea Celular , Supervivencia Celular , Ciclooxigenasa 2/metabolismo , Inhibidores Enzimáticos/metabolismo , Ratones , Palmitatos/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/genética , Propranolol/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Quinacrina/metabolismo , Proteína bcl-X/genética , Receptor fas/metabolismoRESUMEN
Gap-junctional channels, permeable to large hydrophilic solutes of up to M(r) approximately 1,000, are responsible for cell-to-cell communication. Phosphorylation of connexin 43 (Cx43) by PKC abolishes the permeability of gap-junctional channels and hemichannels to large hydrophilic solutes, but not to small inorganic ions. Here, we report on a methodology to produce purified hemichannels of controlled subunit composition and apply it to the generation of hemichannels with variable number of PKC-phosphorylated subunits. The subunit composition was determined by luminescence resonance energy transfer. We show that all Cx43 subunits in the hemichannel hexamer have to be phosphorylated to abolish sucrose (M(r) 342) permeability. We also show that the hemichannel pores with all subunits phosphorylated by PKC have a sizable diameter, allowing for permeation of the small hydrophilic solute ethyleneglycol (M(r) 62). These results indicate that phosphorylation of Cx43 by PKC alters the hemichannel size selectivity and explain why PKC activity affects dye transfer between cells without consistent effects on electrical communication.
Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Tamaño de la Partícula , Proteína Quinasa C/metabolismo , Cromatografía en Gel , Transferencia de Energía , Permeabilidad , Fosforilación , Subunidades de Proteína/metabolismo , Solubilidad , Sacarosa/metabolismo , Terbio/metabolismoRESUMEN
Approximately 25% of all genome coding sequences correspond to membrane proteins, which perform varied and essential functions in cells. Eukaryotic integral membrane proteins are predominantly alpha-helical proteins that span the membrane several times. The most frequent approach to identifying transmembrane-helix amino acids essential for function is to substitute native residues, one at a time, with Cys or Ala (Cys- and Ala-scanning mutagenesis). Here, we present a new approach, in which complete transmembrane-helix native sequences are substituted with poly-Ala sequences. We show that the basic functional features of two dissimilar membrane proteins, which function as a channel and a pump, respectively, are maintained when certain individual alpha-helices are replaced with poly-Ala sequences. This approach ("helix-scanning mutagenesis") allows for rapid identification of helices containing residues essential for function and can be used as a primary helix-screening tool, followed by individual amino acid substitutions when specific helix poly-Ala replacements cause functional changes in the protein.
Asunto(s)
Alanina , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Péptidos , Secuencia de Aminoácidos , Conexina 43/química , Conexina 43/metabolismo , Cisteína , Uniones Comunicantes/fisiología , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutagénesis Sitio-Dirigida , Oocitos/fisiología , Plásmidos , Estructura Secundaria de ProteínaRESUMEN
Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.
Asunto(s)
Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Fosfolipasas de Tipo C/farmacología , 1-Butanol/farmacología , Animales , Hidrocarburos Aromáticos con Puentes/farmacología , Bovinos , Células Cultivadas , Ciclooxigenasa 2 , Inhibidores Enzimáticos/farmacología , Norbornanos , Ácidos Fosfatidicos/farmacología , Fosfolipasa D/farmacología , Propranolol/farmacología , ARN Mensajero/análisis , Tiocarbamatos , Tionas/farmacologíaRESUMEN
A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidylcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.