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To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR â Gαo â Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.
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Membrana Celular/metabolismo , Extensiones de la Superficie Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Aparato de Golgi/metabolismo , Animales , Línea Celular , Drosophila , Femenino , GTP Fosfohidrolasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.
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3-Hidroxiesteroide Deshidrogenasas , Drosophila , Desnutrición , Animales , Drosophila/fisiología , Drosophila melanogaster/fisiología , Ecdisona/genética , Escherichia coli , LarvaRESUMEN
BACKGROUND: Patients carrying pathogenic variants in GNAO1 present a phenotypic spectrum ranging from severe early-onset epileptic encephalopathy and developmental delay to mild adolescent/adult-onset dystonia. Genotype-phenotype correlation and molecular mechanisms underlying the disease remain understudied. METHODS: We analyzed the clinical course of a child carrying the novel GNAO1 mutation c.38T>C;p.Leu13Pro, and structural, biochemical, and cellular properties of the corresponding mutant Gαo-GNAO1-encoded protein-alongside the related mutation c.68T>C;p.Leu23Pro. RESULTS: The main clinical feature was parkinsonism with bradykinesia and rigidity, unlike the hyperkinetic movement disorder commonly associated with GNAO1 mutations. The Leu â Pro substitutions have no impact on enzymatic activity or overall folding of Gαo but uniquely destabilize the N-terminal α-helix, blocking formation of the heterotrimeric G-protein and disabling activation by G-protein-coupled receptors. CONCLUSIONS: Our study defines a parkinsonism phenotype within the spectrum of GNAO1 disorders and suggests a genotype-phenotype correlation by GNAO1 mutations targeting the N-terminal α-helix of Gαo. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Trastornos del Movimiento , Trastornos Parkinsonianos , Adolescente , Niño , Humanos , Estudios de Asociación Genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Trastornos del Movimiento/genética , Mutación/genética , Trastornos Parkinsonianos/genética , Conformación Proteica en Hélice alfaRESUMEN
BACKGROUND: Defects in GNAO1, the gene encoding the major neuronal G-protein Gαo, are related to neurodevelopmental disorders, epilepsy, and movement disorders. Nevertheless, there is a poor understanding of how molecular mechanisms explain the different phenotypes. OBJECTIVES: We aimed to analyze the clinical phenotype and the molecular characterization of GNAO1-related disorders. METHODS: Patients were recruited in collaboration with the Spanish GNAO1 Association. For patient phenotyping, direct clinical evaluation, analysis of homemade-videos, and an online questionnaire completed by families were analyzed. We studied Gαo cellular expression, the interactions of the partner proteins, and binding to guanosine triphosphate (GTP) and G-protein-coupled receptors (GPCRs). RESULTS: Eighteen patients with GNAO1 genetic defects had a complex neurodevelopmental disorder, epilepsy, central hypotonia, and movement disorders. Eleven patients showed neurological deterioration, recurrent hyperkinetic crisis with partial recovery, and secondary complications leading to death in three cases. Deep brain stimulation improved hyperkinetic crisis, but had inconsistent benefits in dystonia. The molecular defects caused by pathogenic Gαo were aberrant GTP binding and hydrolysis activities, an inability to interact with cellular binding partners, and reduced coupling to GPCRs. Decreased localization of Gαo in the plasma membrane was correlated with the phenotype of "developmental and epileptic encephalopathy 17." We observed a genotype-phenotype correlation, pathogenic variants in position 203 were related to developmental and epileptic encephalopathy, whereas those in position 209 were related to neurodevelopmental disorder with involuntary movements. Milder phenotypes were associated with other molecular defects such as del.16q12.2q21 and I344del. CONCLUSION: We highlight the complexity of the motor phenotype, which is characterized by fluctuations throughout the day, and hyperkinetic crisis with a distinct post-hyperkinetic crisis state. We confirm a molecular-based genotype-phenotype correlation for specific variants. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis on G protein α subunits, restricting their activity downstream from G protein-coupled receptors. Here we identify Drosophila Double hit (Dhit) as a dual RGS regulator of Gαo. In addition to the conventional GTPase-activating action, Dhit possesses the guanine nucleotide dissociation inhibitor (GDI) activity, slowing the rate of GTP uptake by Gαo; both activities are mediated by the same RGS domain. These findings are recapitulated using homologous mammalian Gαo/i proteins and RGS19. Crystal structure and mutagenesis studies provide clues into the molecular mechanism for this unprecedented GDI activity. Physiologically, we confirm this activity in Drosophila asymmetric cell divisions and HEK293T cells. We show that the oncogenic Gαo mutant found in breast cancer escapes this GDI regulation. Our studies identify Dhit and its homologs as double-action regulators, inhibiting Gαo/i proteins both through suppression of their activation and acceleration of their inactivation through the single RGS domain.
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Proteínas de Drosophila/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas RGS/metabolismo , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/metabolismo , Cristalografía por Rayos X , Drosophila melanogaster , Femenino , Guanosina Trifosfato/química , Células HEK293 , Humanos , Hidrólisis , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , Transducción de Señal , Factores de TiempoRESUMEN
WNT signaling plays paramount roles in organism development, physiology, and disease, representing a highly attractive target for drug development. However, no WNT-modulating drugs have been approved, with several candidates trudging through the early clinical trials. This delay instigates alternative approaches to discover WNT-modulating drugs. Natural products were the source of therapeutics for centuries, but the chemical diversity they offer, especially when looking at different taxonomic groups and habitats, is still to a large extent unexplored. These considerations urge researchers to screen natural compounds for the WNT-modulatory activities. Since several reviews on such endeavors exist, we here have attempted to present these efforts as "Land and sea tales" (citing the book title by Rudyard Kipling) superimposing them onto the traditional pipeline of drug discovery and early development. In doing so, we illustrate each step of the pipeline with case studies stemming from our own research. It will become obvious that several steps of the pipeline need to be modified when applied to natural products rather than to synthetic libraries. Yet the main message of this chapter is that natural compounds represent a powerful source for the WNT signaling modulators and can be developed towards drug candidates against WNT-dependent maladies.
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Productos Biológicos , Vía de Señalización Wnt , Productos Biológicos/farmacología , Descubrimiento de Drogas , HumanosRESUMEN
The biotransformation of a mixture of resveratrol and pterostilbene was performed by the protein secretome of Botrytis cinerea. Several reaction conditions were tested to overcome solubility issues and to improve enzymatic activity. Using MeOH as cosolvent, a series of unusual methoxylated compounds was generated. The reaction was scaled-up, and the resulting mixture purified by semipreparative HPLC-PDA-ELSD-MS. Using this approach, 15 analogues were isolated in one step. Upon full characterization by NMR and HRMS analyses, eight of the compounds were new. The antibacterial activities of the isolated compounds were evaluated in vitro against the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The selectivity index was calculated based on cytotoxic assays performed against human liver carcinoma cells (HepG2) and the human breast epithelial cell line (MCF10A). Some compounds revealed remarkable antibacterial activity against multidrug-resistant strains of S. aureus with moderate human cell line cytotoxicity.
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Antibacterianos/farmacología , Botrytis/enzimología , Farmacorresistencia Bacteriana/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Estilbenos/farmacología , Biotransformación , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Pruebas de Sensibilidad Microbiana , Prueba de Estudio ConceptualRESUMEN
Triple-negative breast cancer (TNBC) represents the deadliest form of gynecological tumors currently lacking targeted therapies. The ethanol extract of the North Pacific brittle star Ophiura sarsii presented promising anti-TNBC activities. After elimination of the inert material, the active extract was submitted to a bioguided isolation approach using high-resolution semipreparative HPLC-UV, resulting in one-step isolation of an unusual porphyrin derivative possessing strong cytotoxic activity. HRMS and 2D NMR resulted in the structure elucidation of the compound as (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid. Never identified before in Ophiuroidea, porphyrins have found broad applications as photosensitizers in the anticancer photodynamic therapy. The simple isolation of a cytotoxic porphyrin from an abundant brittle star species we describe here may pave the way for novel natural-based developments of targeted anti-cancer therapies.
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Antineoplásicos/farmacología , Organismos Acuáticos/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Estructura Molecular , Fármacos Fotosensibilizantes/aislamiento & purificación , Porfirinas/aislamiento & purificación , Relación Estructura-Actividad , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. Here, we demonstrate that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. We identify Ankyrin 2 as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. Our work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function.
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Ancirinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores Frizzled/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Microtúbulos/metabolismo , Neuronas/citología , Proteína Wnt1/metabolismo , Animales , Línea Celular , Secuencia Conservada , Drosophila melanogaster/citología , Mamíferos , Ratones , Neuritas/metabolismo , Unión Neuromuscular/metabolismo , Neuronas/metabolismo , Unión Proteica , Transducción de Señal , Sinapsis/metabolismoRESUMEN
Overactivation of the Wnt signalling pathway underlies oncogenic transformation and proliferation in many cancers, including the triple-negative breast cancer (TNBC), the deadliest form of tumour in the breast, taking about a quarter of a million lives annually worldwide. No clinically approved targeted therapies attacking Wnt signalling currently exist. Repositioning of approved drugs is a promising approach in drug discovery. In the present study we show that a multi-purpose drug suramin inhibits Wnt signalling and proliferation of TNBC cells in vitro and in mouse models, inhibiting a component in the upper levels of the pathway. Through a set of investigations we identify heterotrimeric G proteins and regulation of Wnt endocytosis as the likely target of suramin in this pathway. G protein-dependent endocytosis of plasma membrane-located components of the Wnt pathway was previously shown to be important for amplification of the signal in this cascade. Our data identify endocytic regulation within Wnt signalling as a promising target for anti-Wnt and anti-cancer drug discovery. Suramin, as the first example of such drug or its analogues might pave the way for the appearance of first-in-class targeted therapies against TNBC and other Wnt-dependent cancers.
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Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Proliferación Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Transducción de Señal/efectos de los fármacos , Suramina/farmacología , Proteína Wnt3A/metabolismo , Animales , Neoplasias de la Mama/patología , Femenino , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Surgical creation of a neovagina using the sigmoid was one of the main techniques used in patients with Mayer-Rokinatsky-Küster-Hauser syndrome. Nowadays, this surgery is not common as a result of the high frequency of complications and adverse outcomes, one of which is sigmoid neovagina prolapse. There are no standards of treatment because of the rarity of these clinical events; therefore, any medical case is important. We present a case report of a 72-year-old patient with prolapse of the sigmoid stump. Perscrutation of this example allows us to conclude that laparoscopic sacrocolpopexy is the optimal operation for patients with apical prolapse and a history of sigmoidal colpopoiesis owing to its high level of safety and excellent outcomes.
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Prolapso de Órgano Pélvico/cirugía , Estructuras Creadas Quirúrgicamente/efectos adversos , Vagina/cirugía , Trastornos del Desarrollo Sexual 46, XX/cirugía , Anciano , Colon Sigmoide/trasplante , Anomalías Congénitas/cirugía , Femenino , Humanos , Laparoscopía , Conductos Paramesonéfricos/anomalías , Conductos Paramesonéfricos/cirugía , Prolapso de Órgano Pélvico/etiologíaRESUMEN
GNAO1 mutated in pediatric encephalopathies encodes the major neuronal G protein Gαo. Of the more than 80 pathogenic mutations, most are single amino acid substitutions spreading across the Gαo sequence. We performed extensive characterization of Gαo mutants, showing abnormal GTP uptake and hydrolysis and deficiencies in binding Gßγ and RGS19. Plasma membrane localization of Gαo was decreased for a subset of mutations that leads to epilepsy; dominant interactions with GPCRs also emerged for the more severe mutants. Pathogenic mutants massively gained interaction with Ric8A and, surprisingly, Ric8B proteins, relocalizing them from cytoplasm to Golgi. Of these 2 mandatory Gα-subunit chaperones, Ric8A is normally responsible for the Gαi/Gαo, Gαq, and Gα12/Gα13 subfamilies, and Ric8B solely responsible for Gαs/Gαolf. Ric8 mediates the disease dominance when engaging in neomorphic interactions with pathogenic Gαo through imbalance of the neuronal G protein signaling networks. As the strength of Gαo-Ric8B interactions correlates with disease severity, our study further identifies an efficient biomarker and predictor for clinical manifestations in GNAO1 encephalopathies. Our work uncovers the neomorphic molecular mechanism of mutations underlying pediatric encephalopathies and offers insights into other maladies caused by G protein malfunctioning and further genetic diseases.
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Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Animales , Femenino , Humanos , Masculino , Encefalopatías/genética , Encefalopatías/metabolismo , Encefalopatías/patología , Drosophila melanogaster , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , MutaciónRESUMEN
BACKGROUND: De novo pathogenic variants in GNAO1-the gene encoding the major neuronal G protein Gαo-cause pediatric encephalopathies and other neurological deficiencies largely refractory to available therapies. Zn2+ emerged to restore guanosine triphosphate hydrolysis and cellular interactions of pathogenic Gαo; dietary zinc salt supplementation improves lifespan and motoric function in a Drosophila disease model. METHODS: Using biochemical, animal, and first-in-human studies, we provide support for the patient stratification and application of zinc acetate in GNAO1-associated disorders. FINDINGS: We show that 16 different pathogenic missense variants cluster in three distinct groups in their responsiveness to Zn2+, and we provide the safety study in a mouse disease model. We further describe treatment of a 3-year-old patient with the common pathogenic GNAO1 variant c607G>A, p.Gly203Arg with oral 50 mg zinc (in the form of zinc acetate) daily, as applied in Wilson's disease. During 11 months of treatment, the patient shows cessation of daily dyskinetic crises, improved Burke-Fahn Marsden Dystonia Rating Scale movement score, reduction in epileptic seizures, and an excellent safety profile. CONCLUSIONS: Our findings warrant a large-scale clinical trial and might set the new standard of care for GNAO1-related disorders. FUNDING: This work was funded by the Russian Science Foundation (grant #21-15-00138) and GNAO1 España.
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In natural products (NPs) research, methods for the efficient prioritization of natural extracts (NEs) are key for discovering novel bioactive NPs. In this study a biodiverse collection of 1,600 NEs, previously analyzed by UHPLC-HRMS2 metabolite profiling was screened for Wnt pathway regulation. The results of the biological screening drove the selection of a subset of 30 non-toxic NEs with an inhibitory IC50 ≤ 5 µg/mL. To increase the chance of finding structurally novel bioactive NPs, Inventa, a computational tool for automated scoring of NEs based on structural novelty was used to mine the HRMS2 analysis and dereplication results. After this, four out of the 30 bioactive NEs were shortlisted by this approach. The most promising sample was the ethyl acetate extract of the leaves of Hymenocardia punctata (Phyllanthaceae). Further phytochemical investigations of this species resulted in the isolation of three known prenylated flavones (3, 5, 7) and ten novel bicyclo[3.3.1]non-3-ene-2,9-diones (1, 2, 4, 6, 8-13), named Hymenotamayonins. Assessment of the Wnt inhibitory activity of these compounds revealed that two prenylated flavones and three novel bicyclic compounds showed interesting activity without apparent cytotoxicity. This study highlights the potential of combining Inventa's structural novelty scores with biological screening results to effectively discover novel bioactive NPs in large NE collections.
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Among different strategies to develop novel therapies, drug repositioning (aka repurposing) aims at identifying new uses of an already approved or investigational drug. This approach has the advantages of availability of the extensive pre-existing knowledge of the drug's safety, pharmacology and toxicology, manufacturing and formulation. It provides advantages to the risk-versus-rewards trade-off as compared to the costly and time-consuming de novo drug discovery process. Clofazimine, a red-colored synthetic derivative of riminophenazines initially isolated from lichens, was first synthesized in the 1950 s, and passed through several phases of repositioning in its history as a drug. Being initially developed as an anti-tuberculosis treatment, it was repurposed for the treatment of leprosy, prior to re-repositioning for the treatment of multidrug-resistant tuberculosis and other infections. Since 1990 s, reports on the anticancer properties of clofazimine, both in vitro and in vivo, started to appear. Among the diverse mechanisms of action proposed, the activity of clofazimine as a specific inhibitor of the oncogenic Wnt signaling pathway has recently emerged as the promising targeting mechanism of the drug against breast, colon, liver, and other forms of cancer. Seventy years after the initial discovery, clofazimine's journey as a drug finding new applications continues, serving as a colorful illustration of drug repurposing in modern pharmacology.
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Clofazimina , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Clofazimina/farmacología , Clofazimina/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Descubrimiento de Drogas , Antituberculosos/farmacología , Antituberculosos/uso terapéuticoRESUMEN
Intracellular signaling orchestrates an organism's development and functioning and underlies various pathologies, such as cancer, when aberrant. A universal cell signaling characteristic is channel capacity - the measure of how much information a given transmitting system can reliably transduce. Here, we describe improved approaches to quantify GPCR signaling channel capacity in single cells, averaged across cell population. We assess the channel capacity based on distribution of residuals by the cellular response amplitude. We further develop means to handle irregularly responding cancer cells using the integral values of their response to different agonist concentrations. These approaches enabled us to analyze, for the first time, channel capacity in single cancer cells. A universal feature emerging for different cancer cell types is a decreased channel capacity of their GPCR signaling. These findings provide experimental validation to the hypothesis that cancer is an information disease, bearing importance for basic cancer biology and anticancer drug discovery.
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The Wnt signaling pathway within host cells regulates infections by several pathogenic bacteria and viruses. Recent studies suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on ß-catenin and can be inhibited by the antileprotic drug clofazimine. Since clofazimine has been identified by us as a specific inhibitor of Wnt/ß-catenin signaling, these works could indicate a potential role of the Wnt pathway in SARS-CoV-2 infection. Here, we show that the Wnt pathway is active in pulmonary epithelial cells. However, we find that in multiple assays, SARS-CoV-2 infection is insensitive to Wnt inhibitors, including clofazimine, acting at different levels within the pathway. Our findings assert that endogenous Wnt signaling in the lung is unlikely required or involved in the SARS-CoV-2 infection and that pharmacological inhibition of this pathway with clofazimine or other compounds is not a universal way to develop treatments against the SARS-CoV-2 infection. IMPORTANCE The development of inhibitors of the SARS-CoV-2 infection remains a need of utmost importance. The Wnt signaling pathway in host cells is often implicated in infections by bacteria and viruses. In this work, we show that, despite previous indications, pharmacological modulation of the Wnt pathway does not represent a promising strategy to control SARS-CoV-2 infection in lung epithelia.
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COVID-19 , Humanos , COVID-19/patología , beta Catenina , Clofazimina , SARS-CoV-2 , Pulmón , Células EpitelialesRESUMEN
BACKGROUND: The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants. METHODS: In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery. FINDINGS: We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gαo. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracellular localization, Gßγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged. CONCLUSIONS: This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this analysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gαo. FUNDING: This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.
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Proteínas de Unión al GTP , Ensayos Analíticos de Alto Rendimiento , Humanos , Niño , Evaluación Preclínica de Medicamentos , Mutación/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismoRESUMEN
A Pacific brittle star Ophiura sarsii has previously been shown to produce a chlorin (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) (1) with potent phototoxic activities, making it applicable to photodynamic therapy. Using extensive LC-MS metabolite profiling, molecular network analysis, and targeted isolation with de novo NMR structure elucidation, we herein identify five additional chlorin compounds from O. sarsii and its deep-sea relative O. ooplax: 10S-Hydroxypheophorbide a (2), Pheophorbide a (3), Pyropheophorbide a (4), (3S,4S,21R)-14-Ethyl-9-(hydroxymethyl)-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (5), and (3S,4S,21R)-14-Ethyl-21-hydroxy-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (6). Chlorins 5 and 6 have not been previously reported in natural sources. Interestingly, low amounts of chlorins 1-4 and 6 could also be identified in a distant species, the basket star Gorgonocephalus cf. eucnemis, demonstrating that chlorins are produced by a wide spectrum of marine invertebrates of the class Ophiuroidea. Following the purification of these major Ophiura chlorin metabolites, we discovered the significant singlet oxygen quantum yield upon their photoinduction and the resulting phototoxicity against triple-negative breast cancer BT-20 cells. These studies identify an arsenal of brittle star chlorins as natural photosensitizers with potential photodynamic therapy applications.