RESUMEN
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.
Asunto(s)
Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/farmacología , COVID-19/inmunología , COVID-19/prevención & control , Pandemias , SARS-CoV-2/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/epidemiología , Vacunas contra la COVID-19/administración & dosificación , Camélidos del Nuevo Mundo/inmunología , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Pandemias/prevención & control , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , SARS-CoV-2/genética , Anticuerpos de Dominio Único/administración & dosificación , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/administración & dosificación , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The mitochondrial pyruvate carrier (MPC) is an inner mitochondrial membrane complex that plays a critical role in intermediary metabolism. Inhibition of the MPC, especially in liver, may have efficacy for treating type 2 diabetes mellitus. Herein, we examined the antidiabetic effects of zaprinast and 7ACC2, small molecules which have been reported to act as MPC inhibitors. Both compounds activated a bioluminescence resonance energy transfer-based MPC reporter assay (reporter sensitive to pyruvate) and potently inhibited pyruvate-mediated respiration in isolated mitochondria. Furthermore, zaprinast and 7ACC2 acutely improved glucose tolerance in diet-induced obese mice in vivo. Although some findings were suggestive of improved insulin sensitivity, hyperinsulinemic-euglycemic clamp studies did not detect enhanced insulin action in response to 7ACC2 treatment. Rather, our data suggest acute glucose-lowering effects of MPC inhibition may be due to suppressed hepatic gluconeogenesis. Finally, we used reporter sensitive to pyruvate to screen a chemical library of drugs and identified 35 potentially novel MPC modulators. Using available evidence, we generated a pharmacophore model to prioritize which hits to pursue. Our analysis revealed carsalam and six quinolone antibiotics, as well as 7ACC1, share a common pharmacophore with 7ACC2. We validated that these compounds are novel inhibitors of the MPC and suppress hepatocyte glucose production and demonstrated that one quinolone (nalidixic acid) improved glucose tolerance in obese mice. In conclusion, these data demonstrate the feasibility of therapeutic targeting of the MPC for treating diabetes and provide scaffolds that can be used to develop potent and novel classes of MPC inhibitors.
Asunto(s)
Proteínas de Transporte de Anión , Proteínas de Transporte de Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos , Obesidad , Quinolonas , Animales , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta , Glucosa/metabolismo , Ratones , Ratones Obesos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Ácido Pirúvico/metabolismo , Quinolonas/farmacologíaRESUMEN
The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening.
Asunto(s)
Antimaláricos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Glucosa/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Antimaláricos/química , Células Cultivadas , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/parasitología , Expresión Génica , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 3/genética , Transportador de Glucosa de Tipo 3/metabolismo , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Especificidad de la Especie , Relación Estructura-Actividad , TritioRESUMEN
Adhesion to and clearance of the mesothelial monolayer are key early events in metastatic seeding of ovarian cancer. ROR2 is a receptor tyrosine kinase that interacts with Wnt5a ligand to activate noncanonical Wnt signaling and has been previously shown to be upregulated in ovarian cancer tissue. However, no prior study has evaluated the mechanistic role of ROR2 in ovarian cancer. Through a cellular high-throughput genetic screen, we independently identified ROR2 as a driver of ovarian tumor cell adhesion and invasion. ROR2 expression in ovarian tumor cells serves to drive directed cell migration preferentially toward areas of high Wnt5a ligand, such as the mesothelial lined omentum. In addition, ROR2 promotes ovarian tumor cell adhesion and clearance of a mesothelial monolayer. Depletion of ROR2, in tumor cells, reduces metastatic tumor burden in a syngeneic model of ovarian cancer. These findings support the role of ROR2 in ovarian tumor cells as a critical factor contributing to the early steps of metastasis. Therapeutic targeting of the ROR2/Wnt5a signaling axis could provide a means of improving treatment for patients with advanced ovarian cancer. IMPLICATIONS: This study demonstrates that ROR2 in ovarian cancer cells is important for directed migration to the metastatic niche and provides a potential signaling axis of interest for therapeutic targeting in ovarian cancer.
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Movimiento Celular , Invasividad Neoplásica , Neoplasias Ováricas , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Proteína Wnt-5a , Femenino , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/genética , Humanos , Ratones , Animales , Línea Celular Tumoral , Vía de Señalización Wnt , Transducción de SeñalRESUMEN
Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders.
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Neuroesteroides , Humanos , Autofagia , Macroautofagia , Riñón , LisosomasRESUMEN
Gamma-secretase is a multiprotein, intramembrane-cleaving protease with a growing list of protein substrates, including the Notch receptors and the amyloid precursor protein. The four components of gamma-secretase complex--presenilin (PS), nicastrin (NCT), Pen2, and Aph1--are all thought to be essential for activity. The catalytic domain resides within PS proteins, NCT has been suggested to be critical for substrate recognition, and the contributions of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by the observation that a critical residue (E332) in NCT, which had been thought to be essential for gamma-secretase activity, is instead involved in complex maturation. Here, we report that NCT is dispensable for gamma-secretase activity. NCT-independent gamma-secretase activity can be detected in two independent NCT-deficient mouse embryonic fibroblast lines and blocked by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and L-685,458. This catalytic activity requires prior ectodomain shedding of the substrate and can cleave ligand-activated endogenous Notch receptors, indicating presence of this activity at the plasma membrane. Small interfering RNA knockdown experiments demonstrated that NCT-independent gamma-secretase activity requires the presence of PS1, Pen2, and Aph1a but can tolerate knockdown of PS2 or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active enzyme, displaying biochemical properties similar to those of gamma-secretase and roughly 50% of its activity when normalized to PS1 N-terminal fragment levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts to stabilize gamma-secretase but is not required for substrate recognition.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Presenilina-1/metabolismo , Receptores Notch/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Células Cultivadas , Reacciones Cruzadas , Endopeptidasas/metabolismo , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Membranas Intracelulares/metabolismo , Proteínas de la Membrana , Ratones , Receptores Notch/genéticaRESUMEN
The role of Notch signaling in general and presenilin in particular was analyzed during mouse somitogenesis. We visualize cyclical production of activated Notch (NICD) and establish that somitogenesis requires less NICD than any other tissue in early mouse embryos. Indeed, formation of cervical somites proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice lacking all presenilin alleles, which have no somites. Since Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites without gamma-secretase, presenilin may have a gamma-secretase-independent role in somitogenesis. Embryos triple homozygous for both presenilin null alleles and a Notch allele that is a poor substrate for presenilin (N1(V-->G)) experience fortuitous cleavage of N1(V-->G) by another protease. This restores NICD, anterior segmentation, and bilateral symmetry but does not rescue rostral/caudal identities. These data clarify multiple roles for Notch signaling during segmentation and suggest that the earliest stages of somitogenesis are regulated by both Notch-dependent and Notch-independent functions of presenilin.
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Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/fisiología , Somitos/citología , Somitos/metabolismo , Factores de Transcripción/fisiología , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Diferenciación Celular , Hibridación in Situ , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Microscopía Electrónica de Rastreo , Fenotipo , Presenilina-1 , Presenilina-2 , Receptor Notch1 , Receptor Notch2 , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune γ-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.
RESUMEN
Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune γ-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARSCoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.
RESUMEN
Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Betacoronavirus/inmunología , Infecciones por Coronavirus/terapia , Peptidil-Dipeptidasa A/inmunología , Neumonía Viral/terapia , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/genética , Betacoronavirus/fisiología , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Proteínas Fluorescentes Verdes/genética , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunización Pasiva , Pruebas de Neutralización , Pandemias , Neumonía Viral/inmunología , Neumonía Viral/virología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Internalización del Virus , Replicación Viral , Sueroterapia para COVID-19RESUMEN
Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformansIMPORTANCECryptococcus neoformans is a fungal pathogen that kills â¼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents.
Asunto(s)
Quitosano/metabolismo , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Indometacina/análogos & derivados , Modelos Biológicos , Morfolinas/farmacología , Transducción de Señal/efectos de los fármacos , Fenómenos Químicos , Descubrimiento de Drogas , Indometacina/química , Indometacina/farmacología , Estructura Molecular , Morfolinas/química , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Although the Plasmodium falciparum hexose transporter PfHT has emerged as a promising target for anti-malarial therapy, previously identified small-molecule inhibitors have lacked promising drug-like structural features necessary for development as clinical therapeutics. Taking advantage of emerging insight into structure/function relationships in homologous facilitative hexose transporters and our novel high throughput screening platform, we investigated the ability of compounds satisfying Lipinksi rules for drug likeness to directly interact and inhibit PfHT. The Maybridge HitFinder chemical library was interrogated by searching for compounds that reduce intracellular glucose by >40% at 10 µM. Testing of initial hits via measurement of 2-deoxyglucose (2-DG) uptake in PfHT over-expressing cell lines identified 6 structurally unique glucose transport inhibitors. WU-1 (3-(2,6-dichlorophenyl)-5-methyl-N-[2-(4-methylbenzenesulfonyl)ethyl]-1,2-oxazole-4-carboxamide) blocked 2-DG uptake (IC50 = 5.8 ± 0.6 µM) with minimal effect on the human orthologue class I (GLUTs 1-4), class II (GLUT8) and class III (GLUT5) facilitative glucose transporters. WU-1 showed comparable potency in blocking 2-DG uptake in freed parasites and inhibiting parasite growth, with an IC50 of 6.1 ± 0.8 µM and EC50 of 5.5 ± 0.6 µM, respectively. WU-1 also directly competed for N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) binding and inhibited the transport of D-glucose with an IC50 of 5.9 ± 0.8 µM in liposomes containing purified PfHT. Kinetic analysis revealed that WU-1 acts as a non-competitive inhibitor of zero-trans D-fructose uptake. Decreased potency for WU-1 and the known endofacial ligand cytochalasin B was observed when PfHT was engineered to contain an N-terminal FLAG tag. This modification resulted in a concomitant increase in affinity for 4,6-O-ethylidene-α-D-glucose, an exofacially directed transport antagonist, but did not alter the Km for 2-DG. Taken together, these data are consistent with a model in which WU-1 binds preferentially to the transporter in an inward open conformation and support the feasibility of developing potent and selective PfHT antagonists as a novel class of anti-malarial drugs.
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Antimaláricos , Proteínas de Transporte de Monosacáridos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias , Antimaláricos/química , Antimaláricos/farmacología , Transporte Biológico Activo/efectos de los fármacos , Glucosa/metabolismo , Células HEK293 , Humanos , Ligandos , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Ingeniería de Proteínas , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Bibliotecas de Moléculas PequeñasRESUMEN
Complex formation between the intracellular domain of the Notch receptor (NICD) and the transcription factor CSL is indispensable for transcriptional activation. To understand how NICD displaces CSL-associated co-repressors, we have quantified the binding of different Notch1 ICD regions to a key interaction domain (the beta trefoil domain, or BTD) of human CSL. Electrophoresis, scattering, and titration calorimetry indicate that NICD and BTD combine to form a 1:1 heterodimer. Neither the Notch1 ankyrin domain (ANK) nor C-terminal region contributes binding energy towards BTD. In contrast, binding energy is attributed largely to a short segment including the conserved WFP sequence motif within the RAM region (the approximately 140 residue polypeptide segment N-terminal to the ANK domain); substitution of this motif substantially reduces affinity. Short (< or =25 residues) WFP-containing peptides encoded by the four mammalian Notch genes have similar affinities to BTD; thus, activity differences between paralogues either result from other regions of NICD and CSL or from differences in interaction with downstream components. The importance of RAM was demonstrated by the ability of a short RAM peptides to dissociate NICD:CSL interaction in cellular lysates. These results support an emerging molecular mechanism for the displacement of co-repressors from DNA-bound CSL by NICD.
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Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Receptores Notch/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Calorimetría , Extractos Celulares , Dimerización , Electroforesis en Gel de Poliacrilamida , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/química , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Notch/química , Homología de Secuencia , Soluciones , TermodinámicaRESUMEN
The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP-VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.
Asunto(s)
Antivirales/farmacología , Filoviridae/fisiología , Nucleoproteínas/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Sitios de Unión/efectos de los fármacos , Secuencia Conservada , Evaluación Preclínica de Medicamentos , Filoviridae/efectos de los fármacos , Filoviridae/genética , Polarización de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Técnicas In Vitro , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Ubiquitylation regulates signaling pathways critical for cancer development and, in many cases, targets proteins for degradation. Here, we report that ubiquitylation by RNF4 stabilizes otherwise short-lived oncogenic transcription factors, including ß-catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein. RNF4 enhances the transcriptional activity of these factors, as well as Wnt- and Notch-dependent gene expression. While RNF4 is a SUMO-targeted ubiquitin ligase, protein stabilization requires the substrate's phosphorylation, rather than SUMOylation, and binding to RNF4's arginine-rich motif domain. Stabilization also involves generation of unusual polyubiquitin chains and docking of RNF4 to chromatin. Biologically, RNF4 enhances the tumor phenotype and is essential for cancer cell survival. High levels of RNF4 mRNA correlate with poor survival of a subgroup of breast cancer patients, and RNF4 protein levels are elevated in 30% of human colon adenocarcinomas. Thus, RNF4-dependent ubiquitylation translates transient phosphorylation signal(s) into long-term protein stabilization, resulting in enhanced oncoprotein activation.
Asunto(s)
Proteínas Nucleares/metabolismo , Oncogenes/fisiología , Estabilidad Proteica , Factores de Transcripción/metabolismo , Humanos , UbiquitinaciónRESUMEN
Upon ligand binding, Notch activation and cleavage culminates in the expression of its target genes. Hence, the use of Notch-responsive promoters to drive reporter gene expression provides a flexible and robust approach for monitoring signaling activity. In this chapter, we present an overview of Notch transcriptional reporter assays and discuss different methods for ligand-independent and ligand-dependent activation of Notch in mammalian cells.
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Receptores Notch/genética , Receptores Notch/metabolismo , Activación Transcripcional , Transfección/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Humanos , Regiones Promotoras GenéticasRESUMEN
Notch activation and cleavage releases the Notch intracellular domain (NICD), which translocates to the nucleus, where it associates with its DNA-binding partner CSL to recruit the coactivator MAML and additional cofactors to ultimately activate target gene expression. Taking advantage of the specific interaction between NICD and these factors, we have developed a luciferase complementation imaging (LCI)-based reporter system to quantitatively monitor Notch activation in real time in live cells. In this chapter, we describe the use of Notch LCI reporters for measuring protein interactions and performing detailed kinetic analyses of receptor activation and its responses to various stimuli.
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Imagen Óptica/métodos , Mapeo de Interacción de Proteínas/métodos , Receptores Notch/análisis , Receptores Notch/metabolismo , Transfección/métodos , Animales , Línea Celular , Genes Reporteros , Humanos , Luciferasas/análisis , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Imagen Molecular/métodos , Receptores Notch/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
NOTCH1, which is frequently mutated in T cell acute lymphoblastic leukemia, has been an elusive therapeutic target. In this issue of Cancer Cell, Roti and colleagues demonstrate that inhibiting SERCA calcium pumps preferentially impairs the maturation of the most common class of oncogenic Notch1 mutants, thus uncovering a potential therapeutic avenue.
RESUMEN
Notch2, but not Notch1, plays indispensable roles in kidney organogenesis, and Notch2 haploinsufficiency is associated with Alagille syndrome. We proposed that proximal nephron fates are regulated by a threshold that requires nearly all available free Notch intracellular domains (NICDs) but could not identify the mechanism that explains why Notch2 (N2) is more important than Notch1 (N1). By generating mice that swap their ICDs, we establish that the overall protein concentration, expression domain, or ICD amino acid composition does not account for the differential requirement of these receptors. Instead, we find that the N2 extracellular domain (NECD) increases Notch protein localization to the cell surface during kidney development and is cleaved more efficiently upon ligand binding. This context-specific asymmetry in NICD release efficiency is further enhanced by Fringe. Our results indicate that an elevated N1 surface level could compensate for the loss of N2 signal in specific cell contexts.
Asunto(s)
Síndrome de Alagille/metabolismo , Riñón/embriología , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch2/metabolismo , Síndrome de Alagille/genética , Síndrome de Alagille/patología , Animales , Células Epiteliales/citología , Células Epiteliales/fisiología , Espacio Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Riñón/citología , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Mutantes , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Nefronas/citología , Nefronas/embriología , Nefronas/metabolismo , Podocitos/citología , Podocitos/fisiología , Estructura Terciaria de Proteína/fisiología , Receptor Notch1/química , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/química , Receptor Notch2/genéticaRESUMEN
The Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system.