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1.
Am J Hum Genet ; 104(2): 203-212, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30612693

RESUMO

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Encéfalo/anormalidades , Encéfalo/metabolismo , Deficiência Intelectual/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Animais , Encéfalo/diagnóstico por imagem , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Criança , Pré-Escolar , Simulação por Computador , Feminino , Humanos , Deficiência Intelectual/diagnóstico por imagem , Locomoção , Lisossomos/metabolismo , Masculino , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Sequenciamento Completo do Exoma , Adulto Jovem
2.
Genetics ; 210(3): 925-946, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30401764

RESUMO

Dense core vesicles (DCVs) can transmit signals by releasing neuropeptides from specialized synaptic regions called active zones. DCVs reach the active zone by motorized transport through a long axon. A reverse motor frequently interrupts progress by taking DCVs in the opposite direction. "Guided transport" refers to the mechanism by which outward movements ultimately dominate to bring DCVs to the synaptic region. After guided transport, DCVs alter their interactions with motors and enter a "captured" state. The mechanisms of guided transport and capture of DCVs are unknown. Here, we discovered two proteins that contribute to both processes in Caenorhabditis elegans SAD kinase and a novel conserved protein we named Sentryn are the first proteins found to promote DCV capture. By imaging DCVs moving in various regions of single identified neurons in living animals, we found that DCV guided transport and capture are linked through SAD kinase, Sentryn, and Liprin-α. These proteins act together to regulate DCV motorized transport in a region-specific manner. Between the cell body and the synaptic region, they promote forward transport. In the synaptic region, where all three proteins are highly enriched at active zones, they promote DCV pausing by inhibiting transport in both directions. These three proteins appear to be part of a special subset of active zone-enriched proteins because other active zone proteins do not share their unique functions.

3.
Genetics ; 210(3): 947-968, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30401765

RESUMO

Synaptic vesicles (SVs) transmit signals by releasing neurotransmitters from specialized synaptic regions of neurons. In the synaptic region, SVs are tightly clustered around small structures called active zones. The motor KIF1A transports SVs outward through axons until they are captured in the synaptic region. This transport must be guided in the forward direction because it is opposed by the dynein motor, which causes SVs to reverse direction multiple times en route. The core synapse stability (CSS) system contributes to both guided transport and capture of SVs. We identified Sentryn as a CSS protein that contributes to the synaptic localization of SVs in Caenorhabditis elegans Like the CSS proteins SAD Kinase and SYD-2 (Liprin-α), Sentryn also prevents dynein-dependent accumulation of lysosomes in dendrites in strains lacking JIP3. Genetic analysis showed that Sentryn and SAD Kinase each have at least one nonoverlapping function for the stable accumulation of SVs at synapses that, when combined with their shared functions, enables most of the functions of SYD-2 (Liprin-α) for capturing SVs. Also like other CSS proteins, Sentryn appears enriched at active zones and contributes to active zone structure, suggesting that it is a novel, conserved active zone protein. Sentryn is recruited to active zones by a process dependent on the active zone-enriched CSS protein SYD-2 (Liprin-α). Our results define a specialized group of active zone enriched proteins that can affect motorized transport throughout the neuron and that have roles in both guided transport and capture of SVs.

4.
Oncotarget ; 9(71): 33577-33588, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30323900

RESUMO

Depletion of BRCA1 protein in mouse mammary glands results in defects in lactational development and increased susceptibility to mammary cancer. Extensive work has focussed on the role of BRCA1 in the normal breast and in the development of breast cancer, the cell of origin for BRCA1 tumours and the protein-coding genes altered in BRCA1 deficient cells. However, the role of non-coding RNAs in BRCA1-deficient cells is poorly understood. To evaluate miRNA expression in BRCA1 deficient mammary cells, RNA sequencing was performed on the mammary glands of Brca1 knockout mice. We identified 140 differentially expressed miRNAs, 9 of which were also differentially expressed in human BRCA1 breast tumours or familial non-BRCA1 patients and during normal gland development. We show that BRCA1 binds to putative cis-elements in promoter regions of the miRNAs with the potential to regulate their expression, and that four miRNAs (miR-29b-1-5p, miR-664, miR-16-2 and miR-744) significantly stratified the overall survival of basal-like tumours. Importantly the prognostic value of miR-29b-1-5p was higher in significance than several commonly used clinical biomarkers. These results emphasise the role of Brca1 in modulating expression of miRNAs and highlights the potential for BRCA1 regulated miRNAs to be informative biomarkers associated with BRCA1 loss and survival in breast cancer.

5.
J Cell Sci ; 131(20)2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30254025

RESUMO

The specific organization of the neuronal microtubule cytoskeleton in axons and dendrites is an evolutionarily conserved determinant of neuronal polarity that allows for selective cargo sorting. However, how dendritic microtubules are organized and whether local differences influence cargo transport remains largely unknown. Here, we use live-cell imaging to systematically probe the microtubule organization in Caenorhabditis elegans neurons, and demonstrate the contribution of distinct mechanisms in the organization of dendritic microtubules. We found that most non-ciliated neurons depend on unc-116 (kinesin-1), unc-33 (CRMP) and unc-44 (ankyrin) for correct microtubule organization and polarized cargo transport, as previously reported. Ciliated neurons and the URX neuron, however, use an additional pathway to nucleate microtubules at the tip of the dendrite, from the base of the cilium in ciliated neurons. Since inhibition of distal microtubule nucleation affects distal dendritic transport, we propose a model in which the presence of a microtubule-organizing center at the dendrite tip ensures correct dendritic cargo transport.

6.
Hum Mutat ; 39(12): 2025-2039, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30204945

RESUMO

The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5' noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C>T and PAX5 binding to BRCA2:c.-296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

8.
Oncotarget ; 8(40): 67203-67217, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28978027

RESUMO

The multi-cancer susceptibility locus at 5p15.33 includes TERT, encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT-CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.

9.
Am J Hum Genet ; 101(2): 255-266, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28777932

RESUMO

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.


Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 11/genética , Ciclina D1/genética , Reparo do DNA/genética , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Elementos Facilitadores Genéticos/genética , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença/genética , Humanos , Células MCF-7 , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Guia/genética , RNA Interferente Pequeno/genética
10.
Oncotarget ; 8(7): 10763-10764, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28099932
11.
Diabetes Technol Ther ; 18(9): 586-93, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27403931

RESUMO

BACKGROUND: Sulfonylureas are widely used to treat type 2 diabetes (T2DM). Although genetic variations are associated with sulfonylurea treatment responses in T2DM patients, whether these variations can be used to predict heterogeneous treatment responses is unclear. In this study, we assessed the potential utility of combining information from multiple variants and phenotypes to predict sulfonylurea response. METHODS: Using data from the "Glibenclamide" arm (365 patients) of the Xiaoke Pill Trial that evaluated the safety and efficacy of sulfonylurea, we identified genetic variants associated with sulfonylurea treatment response, and we explored their ability to predict drug response when combined with phenotype information. RESULTS: The association of 780 single-nucleotide polymorphisms (using Infinium HD iSelect chip) with drug efficacy was evaluated, and four genes identified with drug metabolism (FMO2, FMO3, UGT2B15, and CYP51A1, P < 0.05) were found to be associated with changes in HbA1c. In a clinical model, the baseline values of HbA1c and disposition index (DI) were significantly associated with HbA1c and fasting plasma glucose (FPG) target achievements. Compared with clinical models, the inclusion of genetic markers significantly increased the predictive ability for both HbA1c- and FPG-based outcomes. CONCLUSIONS: Our findings suggest that altered protein function in multiple pathways may cooperatively contribute to the increased discrimination by area under receiver operating curve for T2DM patients, and it may explain, in part, the relationship between inter-individual variability and the sulfonylurea response.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glibureto/uso terapêutico , Hipoglicemiantes/uso terapêutico , Polimorfismo de Nucleotídeo Único , Compostos de Sulfonilureia/uso terapêutico , Idoso , Glicemia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Método Duplo-Cego , Feminino , Glucuronosiltransferase/genética , Humanos , Masculino , Pessoa de Meia-Idade , Oxigenases/genética , Esterol 14-Desmetilase/genética , Falha de Tratamento
12.
Hum Mol Genet ; 25(15): 3269-3283, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27378691

RESUMO

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

13.
Am J Hum Genet ; 98(6): 1159-1169, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27259051

RESUMO

A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer.


Assuntos
Cromossomos Humanos Par 14/genética , Neoplasias do Endométrio/genética , Fosfatidilinositol 3-Quinases/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Uterinas/genética , Fator de Transcrição YY1/metabolismo , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Risco , Transdução de Sinais , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Fator de Transcrição YY1/genética
14.
Hum Mol Genet ; 25(22): 5046-5058, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28171565

RESUMO

Genome-wide association studies (GWAS) have identified markers within the WNT4 region on chromosome 1p36.12 showing consistent and strong association with increasing endometriosis risk. Fine mapping using sequence and imputed genotype data has revealed strong candidates for the causal SNPs within these critical regions; however, the molecular pathogenesis of these SNPs is currently unknown. We used gene expression data collected from whole blood from 862 individuals and endometrial tissue from 136 individuals from independent populations of European descent to examine the mechanism underlying endometriosis susceptibility. Association mapping results from 7,090 individuals (2,594 cases and 4,496 controls) supported rs3820282 as the SNP with the strongest association for endometriosis risk (P = 1.84 × 10−5, OR = 1.244 (1.126-1.375)). SNP rs3820282 is a significant eQTL in whole blood decreasing expression of LINC00339 (also known as HSPC157) and increasing expression of CDC42 (P = 2.0 ×10−54 and 4.5x10−4 respectively). The largest effects were for two LINC00339 probes (P = 2.0 ×10−54; 1.0 × 10−34). The eQTL for LINC00339 was also observed in endometrial tissue (P = 2.4 ×10−8) with the same direction of effect for both whole blood and endometrial tissue. There was no evidence for eQTL effects for WNT4. Chromatin conformation capture provides evidence for risk SNPs interacting with the promoters of both LINC00339 and CDC4 and luciferase reporter assays suggest the risk SNP rs12038474 is located in a transcriptional silencer for CDC42 and the risk allele increases expression of CDC42. However, no effect of rs3820282 was observed in the LINC00339 expression in Ishikawa cells. Taken together, our results suggest that SNPs increasing endometriosis risk in this region act through CDC42, but further functional studies are required to rule out inverse regulation of both LINC00339 and CDC42.


Assuntos
Endometriose/genética , RNA Longo não Codificante/genética , Proteína cdc42 de Ligação ao GTP/genética , Estudos de Casos e Controles , Cromossomos Humanos Par 1 , Endometriose/sangue , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Locos de Características Quantitativas , RNA Longo não Codificante/metabolismo , Fatores de Risco , Proteína Wnt4/genética , Proteína Wnt4/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
15.
Genetics ; 201(1): 91-116, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26354975

RESUMO

The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were ∼3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10- and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Dendritos/metabolismo , Dineínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Sinapses/metabolismo , Transmissão Sináptica , Temperatura Ambiente
16.
Genetics ; 201(1): 117-41, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26354976

RESUMO

The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16's organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(-) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(-) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(-) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16's organelle transport regulatory function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios Motores/metabolismo , Organelas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Axônios/metabolismo , Transporte Biológico Ativo , Proteínas de Caenorhabditis elegans/genética , Quinase 5 Dependente de Ciclina/metabolismo
17.
Am J Hum Genet ; 97(2): 329-36, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26211970

RESUMO

The gene(s) whose expression is regulated by allergy risk variants is unknown for many loci identified through genome-wide association studies. Addressing this knowledge gap might point to new therapeutic targets for allergic disease. The aim of this study was to identify the target gene(s) and the functional variant(s) underlying the association between rs7009110 on chromosome 8q21 and allergies. Eight genes are located within 1 Mb of rs7009110. Multivariate association analysis of publicly available exon expression levels from lymphoblastoid cell lines (LCLs) identified a significant association between rs7009110 and the expression of a single gene, PAG1 (p = 0.0017), 732 kb away. Analysis of histone modifications and DNase I hypersensitive sites in LCLs identified four putative regulatory elements (PREs) in the region. Chromosome conformation capture confirmed that two PREs interacted with the PAG1 promoter, one in allele-specific fashion. To determine whether these PREs were functional, LCLs were transfected with PAG1 promoter-driven luciferase reporter constructs. PRE3 acted as a transcriptional enhancer for PAG1 exclusively when it carried the rs2370615:C allergy predisposing allele, a variant in complete linkage disequilibrium with rs7009110. As such, rs2370615, which overlaps RelA transcription factor (TF) binding in LCLs and was found to disrupt Foxo3a binding to PRE3, represents the putative functional variant in this locus. Our studies suggest that the risk-associated allele of rs2370615 predisposes to allergic disease by increasing PAG1 expression, which might promote B cell activation and have a pro-inflammatory effect. Inhibition of PAG1 expression or function might have therapeutic potential for allergic diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Cromossomos Humanos Par 8/genética , Regulação da Expressão Gênica/genética , Hipersensibilidade/genética , Proteínas de Membrana/genética , Linfócitos B/imunologia , Estudos de Associação Genética , Humanos , Hipersensibilidade/imunologia , Desequilíbrio de Ligação , Luciferases , Ativação Linfocitária/genética , Análise Multivariada , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único/genética , Sequências Reguladoras de Ácido Nucleico/genética
18.
Chem Commun (Camb) ; 51(46): 9499-502, 2015 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-25968110

RESUMO

In this study, we investigated the efficacy of an LNA (locked nucleic acid)-modified DNA aptamer named RNV66 targeting VEGF against various breast cancer cell lines. Our results demonstrate that RNV66 efficiently inhibits breast cancer cell proliferation both in vitro and in vivo. Introduction of LNA nucleotides were crucial for higher efficacy. Furthermore, the binding interaction of RNV66 with VEGF was investigated using molecular dynamic simulations leading to the first computational model of the LNA aptamer-VEGF complex blocking its interaction with VEGF-receptor.


Assuntos
Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Guanina , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos Nus , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oligonucleotídeos/química , Carga Tumoral/efeitos dos fármacos
19.
Nat Commun ; 4: 4999, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25248036

RESUMO

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology.


Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 2/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromossomos Humanos Par 2/metabolismo , Feminino , Predisposição Genética para Doença , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células MCF-7 , Polimorfismo de Nucleotídeo Único
20.
Genetics ; 196(3): 745-65, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24653209

RESUMO

Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Neurônios Motores/metabolismo , Neuropeptídeos/metabolismo , Vesículas Secretórias/metabolismo , Animais , Axônios/diagnóstico por imagem , Axônios/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Códon sem Sentido , Dendritos/diagnóstico por imagem , Dendritos/metabolismo , Exocitose , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Fosfoproteínas/metabolismo , Transporte Proteico , Vesículas Secretórias/ultraestrutura , Ultrassonografia , Proteínas de Transporte Vesicular/metabolismo
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