RESUMEN

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.

Asunto(s)

Proteínas Bacterianas , Legionella pneumophila , Mutagénesis , Mutación Missense , Saccharomyces cerevisiae , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Modelos Moleculares

RESUMEN

The thyroid follicular cells originate from the foregut endoderm and elucidating which genes and signaling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to carry an ENU-based forward mutagenesis screen aiming at identifying genes involved in the development and function of the thyroid follicular cells. ENU is an excellent chemical mutagen due to its high mutation efficiency and an indiscriminate selection of genes. A total of 1606 F2 families from 36 ENU treated founders was raised and embryos from F3 generation were collected at 5dpf to perform the whole embryo in situ hybridization with a cocktail probe of thyroid marker thyroglobulin(tg), pituitary marker thyroid stimulating hormone (tshba) to determine the mutagenic phenotype. Among the 1606 F2 families, 112 F2 mutant families with normal development stages except for thyroid dysfunction were identified and divided into three different groups according to their phenotypic characteristics. Further studies of the mutants are likely to shed more insights into the molecular basis of both the thyroid development and function in the zebrafish and vertebrate.

Asunto(s)

Glándula Tiroides , Pez Cebra , Animales , Pez Cebra/genética , Pruebas Genéticas , Mutación , Mutagénesis

RESUMEN

Epithelial-mesenchymal transition (EMT) is a process of cellular plasticity regulated by complex signaling networks. Under physiological conditions, it plays an important role in wound healing and organ repair. Its importance for human disease is given by its central role in chronic fibroproliferative diseases and cancer, which represent leading causes of death worldwide. In tumors, EMT is involved in primary tumor growth, metastasis and therapy resistance. It is therefore a major requisite to investigate and understand the role of EMT and the mechanisms leading to EMT in order to tackle these diseases therapeutically. Forward genetic screens link genome modifications to phenotypes, and have been successfully employed to identify oncogenes, tumor suppressor genes and genes involved in metastasis or therapy resistance. In particular, transposon-based insertional mutagenesis screens and CRISPR-based screens are versatile and easy-to-use tools applied in recent years to discover and identify novel cancer-related mechanisms. Here, we review the contribution of forward genetic screens to our understanding of how EMT is regulated and how it is involved in various aspects of cancer. Based on the current literature, we propose these methods as additional tools to investigate EMT.

RESUMEN

Congenital disorders of glycosylation (CDG) are a heterogenous group of primarily autosomal recessive mendelian diseases caused by disruptions in the synthesis of lipid-linked oligosaccharides and their transfer to proteins. CDGs usually affect multiple organ systems and vary in presentation, even within families. There is currently no cure, and treatment is aimed at ameliorating symptoms and improving quality of life. Here, we describe a chemically induced mouse mutant, tvrm76, with early-onset photoreceptor degeneration. The recessive mutation was mapped to Chromosome 9 and associated with a missense mutation in the Dpagt1 gene encoding UDP-N-acetyl-D-glucosamine:dolichyl-phosphate N-acetyl-D-glucosaminephosphotransferase (EC 2.7.8.15). The mutation is predicted to cause a substitution of aspartic acid with glycine at residue 166 of DPAGT1. This represents the first viable animal model of a Dpagt1 mutation and a novel phenotype for a CDG. The increased expression of Ddit3, and elevated levels of HSPA5 (BiP) suggest the presence of early-onset endoplasmic reticulum (ER) stress. These changes were associated with the induction of photoreceptor apoptosis in tvrm76 retinas. Mutations in human DPAGT1 cause myasthenic syndrome-13 and severe forms of a congenital disorder of glycosylation Type Ij. In contrast, Dpagt1tvrm76 homozygous mice present with congenital photoreceptor degeneration without overt muscle or muscular junction involvement. Our results suggest the possibility of DPAGT1 mutations in human patients that present primarily with retinitis pigmentosa, with little or no muscle disease. Variants in DPAGT1 should be considered when evaluating cases of non-syndromic retinal degeneration.

Asunto(s)

Trastornos Congénitos de Glicosilación , Enfermedades de la Retina , Acetilglucosamina , Animales , Ácido Aspártico/genética , Trastornos Congénitos de Glicosilación/genética , Glicina/genética , Humanos , Ratones , Debilidad Muscular , Mutación , Mutación Missense , Fosfatos , Calidad de Vida , Uridina Difosfato

RESUMEN

Mutant-specific inhibitors of KRASG12C, such as AMG510 (sotorasib) and MRTX849 (adagrasib), offer the unprecedented opportunity to inhibit KRAS, the most frequently mutated and heretofore undruggable oncoprotein. While clinical data are still limited, on-target mutations in KRASG12C at position 12 and other sites are emerging as major drivers of clinical relapse. We identified additional mutations in KRASG12C that impact inhibitor sensitivity through a saturation mutagenesis screen in the KRASG12C NCI-H358 non­small-cell lung cancer (NSCLC) cell line. We also identified individuals in population genetic databases harboring these resistance mutations in their germline and in tumors, including a subset that co-occur with KRASG12C, indicating that these mutations may preexist in patients treated with KRASG12C inhibitors. Notably, through structural modeling, we found that one such mutation (R68L) interferes with the critical protein­drug interface, conferring resistance to both inhibitors. Finally, we uncovered a mutant (S17E) that demonstrated a strong sensitizing phenotype to both inhibitors. Functional studies suggest that S17E sensitizes KRASG12C cells to KRASG12C inhibition by impacting signaling through PI3K/AKT/mTOR but not the MAPK signaling pathway. Our studies highlight the utility of unbiased mutation profiling to understand the functional consequences of all variants of a disease-causing genetic mutant and predict acquired resistant mutations in the targeted therapeutics.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutagénesis , Mutación , Piperazinas , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas , Pirimidinas

RESUMEN

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM), and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function (LOF) is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 LOF resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Taken together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.

Asunto(s)

Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Hexosaminas , Animales , Vías Biosintéticas , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Glicosilación , Hexosaminas/metabolismo , Ratones

RESUMEN

Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5' splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier.

Asunto(s)

Mutación/genética , Empalme del ARN/genética , Retina/patología , Desprendimiento de Retina/genética , Epitelio Pigmentado de la Retina/patología , Simportadores de Sodio-Bicarbonato/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Desprendimiento de Retina/patología , Tomografía de Coherencia Óptica/métodos

RESUMEN

BACKGROUND: Intragenic modifiers (in-phase, second-site variants) are known to have dramatic effects on clinical outcomes, affecting disease attributes such as severity or age of onset. However, despite their clinical importance, the focus of many genetic screens in model systems is on the discovery of extragenic variants, with many labs still relying upon more traditional methods to identify modifiers. However, traditional methods such as PCR and Sanger sequencing can be time-intensive and do not permit a thorough understanding of the intragenic modifier effects in the context of non-isogenic genomic backgrounds. RESULTS: Here, we apply high throughput approaches to identify and understand intragenic modifiers using Caenorhabditis elegans. Specifically, we applied whole genome sequencing (WGS) to a mutagen-induced forward genetic screen to identify intragenic suppressors of a temperature-sensitive zyg-1(it25) allele in C. elegans. ZYG-1 is a polo kinase that is important for centriole function and cell divisions, and mutations that truncate its human orthologue, PLK4, have been associated with microcephaly. Combining WGS and CRISPR/Cas9, we rapidly identify intragenic modifiers, show that these variants are distributed non-randomly throughout zyg-1 and that genomic context plays an important role on phenotypic outcomes. CONCLUSIONS: Ultimately, our work shows that WGS facilitates high-throughput identification of intragenic modifiers in clinically relevant genes by reducing hands-on research time and overall costs and by allowing thorough understanding of the intragenic phenotypic effects in the context of different genetic backgrounds.

Asunto(s)

Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Alelos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Mutación , Proteínas Quinasas/genética , Secuenciación Completa del Genoma

RESUMEN

BACKGROUND: Eribulin mesylate (eribulin) is an efficient microtubule inhibitor that is used for metastatic breast cancer. However, breast cancer can develop resistance to eribulin. This resistance mechanism needs to be elucidated. METHODS: A transposon mutagenesis screen was conducted using a pPB-SB-CMV-puro-SD plasmid and pCMV-PBase transposase. Viability and cytotoxicity were analyzed by MTT assay and flow cytometry, respectively. Real-time PCR and western blot were used for gene expression analysis. In addition, vivo study was also designed to analyze therapy efficiency. RESULTS: TAB2, which is part of the nuclear factor-kappa B (NF-κB) pathway, was identified as a candidate eribulin-resistant gene. TAB2 down-regulation resulted in significantly lower cell viability and higher cytotoxicity of cells treated with eribulin, while TAB2 up-regulation showed opposite results. Similarly, combination of NF-κB inhibitors [Bay-117082 and QNZ (quinazoline derivative)] with eribulin showed significantly lower cell viability and higher drug cytotoxicity than single agent treatment with eribulin in MDA-MB-231 cells. However, QNZ increased NF-κB activity in MCF7 cells by up-regulating TAB2, which reduced the sensitivity to eribulin. Furthermore, combination of Bay-117082 with eribulin induced greater regression of MDA-MB-231 tumors compared to eribulin monotherapy in vivo. CONCLUSIONS: These results consistently illustrated that TAB2-NF-κB pathway may increases resistance to eribulin in breast cancer models. Moreover, these results support the use of a combination strategy of eribulin with NF-κB inhibitors, and provide evidence that transposon mutagenesis screens are capable of identifying drug-resistant genes.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Furanos/uso terapéutico , Cetonas/uso terapéutico , FN-kappa B/metabolismo , Moduladores de Tubulina/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Regulación hacia Abajo , Femenino , Humanos , Estimación de Kaplan-Meier , Pruebas de Mutagenicidad , Transducción de Señal/efectos de los fármacos

RESUMEN

A better understanding of the molecular mechanisms that control the UCP1 expression in brown and beige adipocytes is essential for us to modulate adipose cell fate and promote thermogenesis, which may provide a therapeutic view for the treatment of obesity and obesity-related diseases. To systematically identify cis-element(s) that transcriptionally regulates Ucp1, we here took advantage of the high-throughput CRIPSR-Cas9 screening system, and performed an in situ saturating mutagenesis screen, by using a customized sgRNA library targeting the ~ 20 kb genomic region near Ucp1. Through the screening, we have identified several genomic loci that may contain key regulatory element for Ucp1 expression in cultured brown and white adipocytes in vitro, and in inguinal white adipose tissue in vivo. Our study highlights a broadly useful approach for studying cis-regulatory elements in a high-throughput manner.

RESUMEN

TASK-3 potassium (K+) channels are highly expressed in the central nervous system, regulating the membrane potential of excitable cells. TASK-3 is involved in neurotransmitter action and has been identified as an oncogenic K+ channel. For this reason, the understanding of the action mechanism of pharmacological modulators of these channels is essential to obtain new therapeutic strategies. In this study we describe the binding mode of the potent antagonist PK-THPP into the TASK-3 channel. PK-THPP blocks TASK-1, the closest relative channel of TASK-3, with almost nine-times less potency. Our results confirm that the binding is influenced by the fenestrations state of TASK-3 channels and occurs when they are open. The binding is mainly governed by hydrophobic contacts between the blocker and the residues of the binding site. These interactions occur not only for PK-THPP, but also for the antagonist series based on 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine scaffold (THPP series). However, the marked difference in the potency of THPP series compounds such as 20b, 21, 22 and 23 (PK-THPP) respect to compounds such as 17b, inhibiting TASK-3 channels in the micromolar range is due to the presence of a hydrogen bond acceptor group that can establish interactions with the threonines of the selectivity filter.

Asunto(s)

Simulación del Acoplamiento Molecular , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/química , Piridinas/farmacología , Pirimidinas/farmacología , Animales , Sitios de Unión , Humanos , Bloqueadores de los Canales de Potasio/química , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Unión Proteica , Piridinas/química , Pirimidinas/química , Xenopus

RESUMEN

Chronic fatigue is a debilitating disorder with widespread consequences, but effective treatment strategies are lacking. Novel genetic mouse models of fatigue may prove invaluable for studying its underlying physiological mechanisms and for testing treatments and interventions. In a screen of voluntary wheel-running behavior in N-ethyl-N-nitrosourea mutagenized C57BL/6J mice, we discovered two lines with low body weights and aberrant wheel-running patterns suggestive of a fatigue phenotype. Affected progeny from these lines had lower daily activity levels and exhibited low amplitude circadian rhythm alterations. Their aberrant behavior was characterized by frequent interruptions and periods of inactivity throughout the dark phase of the light-dark cycle and increased levels of activity during the rest or light phase. Expression of the behavioral phenotypes in offspring of strategic crosses was consistent with a recessive inheritance pattern. Mapping of phenotypic abnormalities showed linkage with a single locus on chromosome 1, and whole exome sequencing identified a single point mutation in the Slc2a4 gene encoding the GLUT4 insulin-responsive glucose transporter. The single nucleotide change (A-T, which we named "twiggy") was in the distal end of exon 10 and resulted in a premature stop (Y440*). Additional metabolic phenotyping confirmed that these mice recapitulate phenotypes found in GLUT4 knockout mice. However, to the best of our knowledge, this is the first time a mutation in this gene has been shown to result in extensive changes in general behavioral patterns. These findings suggest that GLUT4 may be involved in circadian behavioral abnormalities and could provide insights into fatigue in humans.

Asunto(s)

Ritmo Circadiano , Codón sin Sentido , Fatiga/genética , Transportador de Glucosa de Tipo 4/genética , Animales , Conducta Animal , Modelos Animales de Enfermedad , Fatiga/fisiopatología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo

RESUMEN

Given attention to both contraception and treatment of infertility, there is a need to identify genes and sequence variants required for mammalian fertility. Recent unbiased mutagenesis strategies have expanded horizons of genetic control of reproduction. Here we show that male mice homozygous for the ethyl-nitroso-urea-induced ferf1 (fertilization failure 1) mutation are infertile, producing apparently normal sperm that does not fertilize oocytes in standard fertilization in vitro fertilization assays. The ferf1 mutation is a single-base change in the Dnah1 gene, encoding an axoneme-associated dynein heavy chain, and previously associated with male infertility in both mice and humans. This missense mutation causes a single-amino-acid change in the DNAH1 protein in ferf1 mutant mice that leads to abnormal sperm clumping, aberrant sperm motility, and the inability of sperm to penetrate the oocyte's zona pellucida; however, the ferf1 mutant sperm is competent to fertilize zona-free oocytes. Taken together, the various mutations affecting the DNAH1 protein in both mouse and human produce a diversity of phenotypes with both subtle and considerable differences. Thus, future identification of the interacting partners of DNAH1 might lead to understanding its unique function among the sperm dyneins.

Asunto(s)

Dineínas , Infertilidad Masculina , Mutación , Oocitos , Motilidad Espermática/genética , Espermatozoides , Animales , Dineínas/genética , Dineínas/metabolismo , Femenino , Fertilización In Vitro , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Mutantes , Oocitos/metabolismo , Oocitos/ultraestructura , Espermatozoides/metabolismo , Espermatozoides/ultraestructura

RESUMEN

Prostate cancer (PC) is the second leading cause of cancer-related deaths in US men, and progression to androgen-independent PC (AIPC) typically results in metastasis and is lethal. However, the mechanisms whereby PC progresses from androgen dependence to androgen independence are not completely understood. Mutagenesis screens to identify novel genes involved in the progression to AIPC have been performed using replication-incompetent lentiviral vectors (LVs). In this approach the LV acts both as a mutagen and as molecular tag to identify nearby genes that may have been dysregulated by the vector provirus, and are candidate AIPC genes. Here we describe protocols for generation of replication-incompetent LV preparations and performing a mutagenesis screen to identify AIPC genes in vitro.

Asunto(s)

Andrógenos/metabolismo , Genes Relacionados con las Neoplasias , Lentivirus/genética , Mutagénesis , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Replicación Viral , Progresión de la Enfermedad , Vectores Genéticos , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Células Tumorales Cultivadas

RESUMEN

Replication-incompetent gammaretroviral (γRV) and lentiviral (LV) vectors have both been used in insertional mutagenesis screens to identify cancer drivers. In this approach the vectors stably integrate in the host cell genome and induce cancers by dysregulating nearby genes. The cells that contain a retroviral vector provirus in or near a proto-oncogene or tumor suppressor are preferentially enriched in a tumor. γRV and LV vectors have different integration profiles and genotoxic potential, making them potentially complementary tools for insertional mutagenesis screens. We performed screens using both γRV and LV vectors to identify driver genes that mediate progression of androgen-independent prostate cancer (AIPC) using a xenotransplant mouse model. Vector transduced LNCaP cells were injected orthotopically into the prostate gland of immunodeficient mice. Mice that developed tumors were castrated to create an androgen-deficient environment and metastatic tumors that developed were analyzed. A high-throughput modified genomic sequencing PCR (MGS-PCR) approach identified the positions of vector integrations in these metastatic tumors. OR2A14, FER1L6, TAOK3, MAN1A2, MBNL2, SERBP1, PLEKHA2, SPTAN1, ADAMTS1, SLC30A5, ABCC1, SLC7A1 and SLC25A24 were identified as candidate prostate cancer (PC) progression genes. TAOK3 and ABCC1 expression in PC patients predicted the risk of recurrence after androgen deprivation therapy. Our data shows that γRV and LV vectors are complementary approaches to identify cancer driver genes which may be promising potential biomarkers and therapeutic targets.

RESUMEN

In an attempt to define novel genetic loci involved in the pathophysiology of primary aldosteronism, a mutagenesis screen after treatment with the alkylating agent N-ethyl-N-nitrosourea was established for the parameter aldosterone. One of the generated mouse lines with hyperaldosteronism was phenotypically and genetically characterized. This mouse line had high aldosterone levels but normal creatinine and urea values. The steroidogenic enzyme expression levels in the adrenal gland did not differ significantly among phenotypically affected and unaffected mice. Upon exome sequencing, point mutations were identified in seven candidate genes (Sspo, Dguok, Hoxaas2, Clstn3, Atm, Tipin and Mapk6). Subsequently, animals were stratified into wild-type and mutated groups according to their genotype for each of these candidate genes. A correlation of their genotypes with the respective aldosterone, aldosterone-to-renin ratio (ARR), urea and creatinine values as well as steroidogenic enzyme expression levels was performed. Aldosterone values were significantly higher in animals carrying mutations in four different genes (Sspo, Dguok, Hoxaas2 and Clstn3) and associated statistically significant adrenal Cyp11b2 overexpression as well as increased ARR was present only in mice with Sspo mutation. In contrast, mutations of the remaining candidate genes (Atm, Tipin and Mapk6) were associated with lower aldosterone values and lower Hsd3b6 expression levels. In summary, these data demonstrate association between the genes Sspo, Dguok, Hoxaas2 and Clstn3 and hyperaldosteronism. Final proofs for the causative nature of the mutations have to come from knock-out and knock-in experiments.

Asunto(s)

Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Aldosterona/sangre , Aldosterona/metabolismo , Animales , Biomarcadores , Modelos Animales de Enfermedad , Exoma , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones , Mutación , Linaje , Polimorfismo de Nucleótido Simple

RESUMEN

Breast cancer (BC) is the second leading cause of malignancy among U.S. women. Metastasis results in a poor prognosis and increased mortality, but the molecular mechanisms by which metastatic tumors occur are not well understood. Identifying the genes that drive the metastatic process could provide targets for improved therapy and biomarkers to improve BC patient outcomes. Using a forward mutagenesis screen, BC cells mutagenized with a replication-incompetent gammaretroviral vector (γRV) were xenotransplanted into the mammary fat pad of immunodeficient mice. In this approach the vector provirus dysregulates nearby genes, providing a selective advantage to transduced cells to form metastases. Metastatic tumors were analyzed for proviral integration sites to identify nearby candidate metastasis genes. The γRV has a transgene cassette that allows for rescue in bacteria and rapid identification of vector integration sites. Using this approach, we identified the previously described metastasis gene WWTR1 (TAZ), and three other novel candidate metastasis genes including SHARPIN. SHARPIN was independently validated in vivo as a BC metastasis gene. Analysis of patient data showed that SHARPIN expression predicts metastasis-free survival after adjuvant therapy. Our approach has broad potential to identify genes involved in oncogenic processes for BC and other cancers. We show here it can identify both known (WWTR1) and novel (SHARPIN) BC metastasis genes.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Gammaretrovirus/genética , Mutagénesis Insercional , Ubiquitinas/genética , Animales , Secuencia de Bases , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Estimación de Kaplan-Meier , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Pronóstico , Interferencia de ARN , Tratamiento con ARN de Interferencia/métodos , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Ensayos Antitumor por Modelo de Xenoinjerto/métodos

RESUMEN

Using a novel retroviral shuttle vector approach we identified genes that collaborate with a patient derived RUNX1 (AML1) mutant. RUNX1 mutations occurs in 40% of myelodysplastic syndromes (MDS). MDS are a group of hematopoietic stem cell disorders that are characterized by dysplasia that often progress to acute myeloid leukemia (AML). Our goal was to identify genes dysregulated by vector-mediated genotoxicity that may collaborate with the RUNX1 mutant (D171N). D171N expressing cells have a survival and engraftment disadvantage and require additional genetic lesions to survive and persist. By dysregulating genes near the integrated vector provirus, the shuttle vector can promote transformation of D171N cells and tag the nearby genes that collaborate with D171N. In our approach, a gammaretroviral shuttle vector that expresses D171N is used to transduce CD105+, Sca-1+ mouse bone marrow. Mutagenized cells are expanded in liquid culture and vector integration sites from surviving cells are then identified using a retroviral shuttle vector approach. We repeatedly recovered integrated vector proviruses near genes (Itpkb, Ccdc12, and Nbeal2). To assess the prognostic significance of the genes identified we examined differential expression, overall survival, and relapse free survival of AML patients with alteration in the genes identified using The Cancer Genome Atlas (TCGA) AML data set. We found that ITPKB functions as an independent factor for poor prognoses and RUNX1 mutations in conjunction with ITPKB, CCDC12, and NBEAL2 have prognostic potential in AML.

Asunto(s)

Carcinogénesis/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Vectores Genéticos/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Animales , Proteínas Sanguíneas/genética , Línea Celular Tumoral , Células HEK293 , Humanos , Leucemia Mieloide Aguda/mortalidad , Ratones , Síndromes Mielodisplásicos/mortalidad , Síndromes Mielodisplásicos/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas/genética , Retroviridae/genética , Integración Viral/genética

RESUMEN

Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. NRC1 (NB-LRR Required for HR-Associated Cell Death-1) is a tomato (Solanum lycopersicum) NB-LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae. To identify mutations in NRC1 that cause increased signalling activity, we generated a random library of NRC1 variants mutated in their nucleotide-binding domain and screened them for the ability to induce an elicitor-independent HR in Nicotiana tabacum. Screening of 1920 clones retrieved 11 gain-of-function mutants, with 10 of them caused by a single amino acid substitution. All substitutions are located in or very close to highly conserved motifs within the nucleotide-binding domain, suggesting modulation of the signalling activity of NRC1. Three-dimensional modelling of the nucleotide-binding domain of NRC1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain-of-function mutations in NRC1 and provides insight into how the activity of this NB-LRR is regulated.

Asunto(s)

Resistencia a la Enfermedad/genética , Mutación , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas/genética , Solanaceae/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Muerte Celular , Cladosporium/metabolismo , Cladosporium/patogenicidad , Genes de Plantas , Leucina/metabolismo , Proteínas Repetidas Ricas en Leucina , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Estructura Molecular , Mutagénesis , Nucleótidos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas/metabolismo , Transducción de Señal , Solanaceae/metabolismo , Solanaceae/microbiología , Nicotiana/genética , Nicotiana/microbiología , Verticillium/metabolismo , Verticillium/patogenicidad

RESUMEN

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia-related genes from many large-scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs.

Asunto(s)

Cilios/patología , Anomalías Congénitas/genética , Modelos Animales de Enfermedad , Animales , Encéfalo/anomalías , Anomalías Congénitas/patología , Discinesias/genética , Discinesias/patología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Mutación , Orgánulos/patología , Transducción de Señal