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1.
Cell ; 157(2): 447-458, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24725410

RESUMO

Maintenance of a constant cell volume in response to extracellular or intracellular osmotic changes is critical for cellular homeostasis. Activation of a ubiquitous volume-regulated anion channel (VRAC) plays a key role in this process; however, its molecular identity in vertebrates remains unknown. Here, we used a cell-based fluorescence assay and performed a genome-wide RNAi screen to find components of VRAC. We identified SWELL1 (LRRC8A), a member of a four-transmembrane protein family with unknown function, as essential for hypotonicity-induced iodide influx. SWELL1 is localized to the plasma membrane, and its knockdown dramatically reduces endogenous VRAC currents and regulatory cell volume decrease in various cell types. Furthermore, point mutations in SWELL1 cause a significant change in VRAC anion selectivity, demonstrating that SWELL1 is an essential VRAC component. These findings enable further molecular characterization of the VRAC channel complex and genetic studies for understanding the function of VRAC in normal physiology and disease.


Assuntos
Tamanho Celular , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Células HEK293 , Células HeLa , Humanos , Iodetos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Interferência de RNA
2.
Cell ; 135(1): 49-60, 2008 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-18854154

RESUMO

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas/metabolismo , Replicação Viral , Linhagem Celular , Humanos , Interferência de RNA , Técnicas do Sistema de Duplo-Híbrido
3.
Nature ; 463(7282): 813-7, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-20027183

RESUMO

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.


Assuntos
Fatores Biológicos/genética , Fatores Biológicos/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Influenza A/crescimento & desenvolvimento , Influenza Humana/genética , Influenza Humana/virologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Biblioteca Gênica , Genoma Humano/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A/classificação , Interferência de RNA , Células Vero , Internalização do Vírus
4.
Int J Pharm ; 663: 124555, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-39111354

RESUMO

This study aimed to investigate the amorphous stabilization of BCS Class II drugs using mesoporous silica as a carrier to produce amorphous solid dispersions. Ibuprofen, fenofibrate, and budesonide were selected as model drugs to evaluate the impact of molecular weight and partition coefficient on the solid state of drug-loaded mesoporous silica (MS) particles. The model drugs were loaded into three grades of MS, SYLYSIA SY730, SYLYSIA SY430, and SYLYSIA SY350, with pore diameters of 2.5 nm, 17 nm, and 21 nm, respectively, at 1:1, 2:1, and 3:1, carrier to drug ratios, and three different loading concentrations using solvent immersion and spray drying techniques. Differential scanning calorimetry (DSC) thermograms of SY430 and SY350 samples exhibited melting point depressions indicating constricted crystallization inside the pores, whereas SY730 samples with melting points matching the pure API may be a result of surface crystallization. Powder x-ray diffraction (PXRD) diffractograms showed all crystalline samples matched the diffraction patterns of the pure API indicating no polymorphic transitions and all 3:1 ratio samples exhibited amorphous halo profiles. Response surface regression analysis and Classification and Regression Tree (CART) analysis suggest carrier to drug ratios, followed by molecular weight, have the most significant impact on the crystallinity of a drug loaded into MS particles.

5.
Proc Natl Acad Sci U S A ; 106(40): 17025-30, 2009 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-19805117

RESUMO

Malignant melanoma is the most aggressive form of cutaneous carcinoma, accounting for 75% of all deaths caused by skin cancers. Microphthalmia-associated transcription factor (MITF) is a master gene regulating melanocyte development and functions as a "lineage addiction" oncogene in malignant melanoma. We have identified the receptor protein tyrosine kinase TYRO3 as an upstream regulator of MITF expression by a genome-wide gain-of-function cDNA screen and show that TYRO3 induces MITF-M expression in a SOX10-dependent manner in melanoma cells. Expression of TYRO3 is significantly elevated in human primary melanoma tissue samples and melanoma cell lines and correlates with MITF-M mRNA levels. TYRO3 overexpression bypasses BRAF(V600E)-induced senescence in primary melanocytes, inducing transformation of non-tumorigenic cell lines. Furthermore, TYRO3 knockdown represses cellular proliferation and colony formation in melanoma cells, and sensitizes them to chemotherapeutic agent-induced apoptosis; TYRO3 knockdown in melanoma cells also inhibits tumorigenesis in vivo. Taken together, these data indicate that TYRO3 may serve as a target for the development of therapeutic agents for melanoma.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Fator de Transcrição Associado à Microftalmia/genética , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Oncotarget ; 8(59): 99913-99930, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29245949

RESUMO

Recent advances in chemotherapeutics highlight the importance of molecularly-targeted perturbagens. Although these therapies typically address dysregulated cancer cell proteins, there are increasing therapeutic modalities that take into consideration cancer cell-extrinsic factors. Targeting components of tumor stroma such as vascular or immune cells has been shown to represent an efficacious approach in cancer treatment. Cancer-associated fibroblasts (CAFs) exemplify an important stromal component that can be exploited in targeted therapeutics, though their employment in drug discovery campaigns has been relatively minimal due to technical logistics in assaying for CAF-tumor interactions. Here we report a 3-dimensional multi-culture tumor:CAF spheroid phenotypic screening platform that can be applied to high-content drug discovery initiatives. Using a functional genomics approach we systematically profiled 1,024 candidate genes for CAF-intrinsic anti-spheroid activity; identifying several CAF genes important for development and maintenance of tumor:CAF co-culture spheroids. Along with previously reported genes such as WNT, we identify CAF-derived targets such as ARAF and COL3A1 upon which the tumor compartment depends for spheroid development. Specifically, we highlight the G-protein-coupled receptor OGR1 as a unique CAF-specific protein that may represent an attractive drug target for treating colorectal cancer. In vivo, murine colon tumor implants in OGR1 knockout mice displayed delayed tumor growth compared to tumors implanted in wild type littermate controls. These findings demonstrate a robust microphysiological screening approach for identifying new CAF targets that may be applied to drug discovery efforts.

7.
Methods Enzymol ; 414: 530-65, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17110210

RESUMO

Recent advances in functional genomics have enabled genome-wide genetic studies in mammalian cells. These include the establishment of high-throughput transfection and viral propagation methodologies, the production of large-scale cDNA and siRNA libraries, and the development of sensitive assay detection processes and instrumentation. The latter has been significantly facilitated by the implementation of automated microscopy and quantitative image analysis, collectively referred to as high-content screening (HCS), toward cell-based functional genomics application. This technology can be applied to whole genome analysis of discrete molecular and phenotypic events at the level of individual cells and promises to significantly expand the scope of functional genomic analyses in mammalian cells. This chapter provides a comprehensive guide for curating and preparing function genomics libraries and performing HCS at the level of the genome.


Assuntos
Biblioteca Genômica , Genômica/instrumentação , Genômica/métodos , Animais , Automação , Biomarcadores/química , Separação Celular , DNA Complementar/metabolismo , Citometria de Fluxo , Biblioteca Gênica , Genoma Humano , Humanos , Microscopia de Fluorescência/métodos , Plasmídeos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
8.
J Biomol Screen ; 20(6): 760-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25616511

RESUMO

Friedreich's ataxia is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin. This deficiency results from expansion of a trinucleotide repeat in the first intron of the frataxin gene. Because this repeat expansion resides in an intron and hence does not alter the amino acid sequence of the frataxin protein, gene reactivation could be of therapeutic benefit. High-throughput screening for frataxin activators has so far met with limited success because current cellular models may not accurately assess endogenous frataxin gene regulation. Here we report the design and validation of genome-engineering tools that enable the generation of human cell lines that express the frataxin gene fused to a luciferase reporter gene from its endogenous locus. Performing a pilot high-throughput genomic screen in a newly established reporter cell line, we uncovered novel negative regulators of frataxin expression. Rational design of small-molecule inhibitors of the identified frataxin repressors and/or high-throughput screening of large siRNA or compound libraries with our system may yield treatments for Friedreich's ataxia.


Assuntos
Descoberta de Drogas , Ataxia de Friedreich/genética , Expressão Gênica , Genes Reporter , Engenharia Genética , Linhagem Celular Transformada , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/terapia , Ensaios de Triagem em Larga Escala , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Dedos de Zinco/genética
9.
PLoS One ; 4(12): e8348, 2009 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20020055

RESUMO

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened approximately 10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.


Assuntos
Distrofina/genética , Éxons/genética , Ensaios de Triagem em Larga Escala/métodos , Oligonucleotídeos Antissenso/farmacologia , Bibliotecas de Moléculas Pequenas/análise , Processamento Alternativo/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Ensaios Enzimáticos , Genes Reporter , Genoma Humano/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Luciferases/metabolismo , Camundongos , Índice Mitótico , Distrofia Muscular de Duchenne/genética , Fosfotransferases/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologia , Moduladores de Tubulina/farmacologia
10.
Nat Methods ; 4(10): 847-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17828270

RESUMO

We describe a statistical analysis methodology designed to minimize the impact of off-target activities upon large-scale RNA interference (RNAi) screens in mammalian cells. Application of this approach enhances reconfirmation rates and facilitates the experimental validation of new gene activities through the probability-based identification of multiple distinct and active small interfering RNAs (siRNAs) targeting the same gene. We further extend this approach to establish that the optimal redundancy for efficacious RNAi collections is between 4-6 siRNAs per gene.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Probabilidade
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