RESUMEN
CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.
Asunto(s)
Citocromo P-450 CYP2C9/metabolismo , Mutación Missense , Medicamentos bajo Prescripción/metabolismo , Saccharomyces cerevisiae/enzimología , Xenobióticos/metabolismo , Sitios de Unión , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/genética , Pruebas de Enzimas , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fenitoína/química , Polimorfismo Genético , Medicamentos bajo Prescripción/química , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Transgenes , Warfarina/química , Warfarina/metabolismo , Xenobióticos/químicaRESUMEN
Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.
Asunto(s)
Dominio Catalítico , Variación Genética , Mutación Missense , Vitamina K Epóxido Reductasas/química , Vitamina K Epóxido Reductasas/genética , Cisteína/química , Resistencia a Medicamentos , Células HEK293 , Humanos , Errores Innatos del Metabolismo , Modelos Moleculares , Análisis de Secuencia de ADN , Warfarina/farmacologíaRESUMEN
Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.
Asunto(s)
Bioensayo , Biblioteca de Genes , Plásmidos/química , Transgenes , Animales , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HT29 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Células 3T3 NIH , Oncogenes , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinasas/genética , Recombinasas/metabolismo , Recombinación Genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Fluorescente RojaAsunto(s)
Reacción en Cadena de la Polimerasa Multiplex/métodos , Variantes Farmacogenómicas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Farmacogenética/métodos , Medicina de Precisión/métodos , Utilización de Procedimientos y Técnicas , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodosRESUMEN
Determining the pathogenicity of genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes requires generalizable, scalable assays. We describe variant abundance by massively parallel sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance simultaneously. We apply VAMP-seq to quantify the abundance of 7,801 single-amino-acid variants of PTEN and TPMT, proteins in which functional variants are clinically actionable. We identify 1,138 PTEN and 777 TPMT variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and show that p.Pro38Ser, which accounts for ~10% of PTEN missense variants in melanoma, functions via a dominant-negative mechanism. Finally, we demonstrate that VAMP-seq is applicable to other genes, highlighting its generalizability.
Asunto(s)
Mutación Missense , Aminoácidos/genética , Línea Celular , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Fosfohidrolasa PTEN/genética , Análisis de Secuencia de ADN/métodosRESUMEN
We found significantly higher incidence of Toxoplasma gondii DNA in eye bank specimens from Joinville in southern Brazil (13/15, 87%) than in São Paulo (3/42, 7%; p = 2.1 × 10E-8). PCR DNA sequence analysis was more sensitive at locus NTS2 than at locus B1; a high frequency of mixed co-infections was detected.
Asunto(s)
Sitios Genéticos , Retina/parasitología , Toxoplasma/aislamiento & purificación , Toxoplasmosis Ocular/epidemiología , Adulto , Anciano , Brasil/epidemiología , Coriorretinitis , Bancos de Ojos , Genotipo , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Prevalencia , Retina/patología , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis Ocular/diagnóstico , Toxoplasmosis Ocular/parasitología , Toxoplasmosis Ocular/patologíaRESUMEN
UNLABELLED: Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE: Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals-including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.
Asunto(s)
Genoma de Protozoos , Sarcocystis/crecimiento & desarrollo , Sarcocystis/genética , Sarcocistosis/parasitología , Sarcocistosis/veterinaria , Animales , Humanos , Estadios del Ciclo de Vida , Filogenia , Proteínas Protozoarias/genética , Sarcocystis/clasificación , Sarcocystis/metabolismoRESUMEN
Increasingly, metabolic potential is proving to be a critical determinant governing a pathogen's virulence as well as its capacity to expand its host range. To understand the potential contribution of metabolism to strain-specific infectivity differences, we present a constraint-based metabolic model of the opportunistic parasite, Toxoplasma gondii. Dominated by three clonal strains (Type I, II, and III demonstrating distinct virulence profiles), T. gondii exhibits a remarkably broad host range. Integrating functional genomic data, our model (which we term as iCS382) reveals that observed strain-specific differences in growth rates are driven by altered capacities for energy production. We further predict strain-specific differences in drug susceptibilities and validate one of these predictions in a drug-based assay, with a Type I strain demonstrating resistance to inhibitors that are effective against a Type II strain. We propose that these observed differences reflect an evolutionary strategy that allows the parasite to extend its host range, as well as result in a subsequent partitioning into discrete strains that display altered virulence profiles across different hosts, different organs, and even cell types.
Asunto(s)
Fibroblastos/parasitología , Regulación de la Expresión Génica , Redes y Vías Metabólicas , Toxoplasma/metabolismo , Toxoplasma/patogenicidad , Antiprotozoarios/farmacología , Difosfonatos/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Fibroblastos/citología , Especificidad del Huésped , Interacciones Huésped-Parásitos , Humanos , Ingeniería Metabólica , Modelos Genéticos , Quinolinas/farmacología , Especificidad de la Especie , Toxoplasma/efectos de los fármacos , Toxoplasma/genética , VirulenciaRESUMEN
A retrospective analysis of 844 IVF-ET cycles demonstrated that changes in E(2) levels after administration of hCG do not influence fertilization, implantation, pregnancy, or live-birth rates. In vitro fertilization cycles with a declining E(2) level have comparable success to those with no change or increasing E(2) levels and should proceed to oocyte retrieval.