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1.
Nature ; 571(7765): 413-418, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31243372

RESUMO

ABTRACT: Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland1,2. FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours3-8. However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive9-11 and oncogenic12-14 roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and-through TLE3 inactivation-promote metastasis driven by the WNT pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element-herein denoted FOXA1 mastermind (FOXMIND)-to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies.


Assuntos
Fator 3-alfa Nuclear de Hepatócito/genética , Mutação/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Fator 3-alfa Nuclear de Hepatócito/química , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Modelos Moleculares , Metástase Neoplásica/genética , Domínios Proteicos , Receptores Androgênicos/metabolismo , Via de Sinalização Wnt
2.
Mol Cell ; 74(3): 521-533.e6, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30952514

RESUMO

Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.


Assuntos
MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Grânulos Citoplasmáticos/genética , Inativação Gênica , Células HeLa , Humanos , Processamento Pós-Transcricional do RNA/genética , RNA não Traduzido/genética , Imagem Individual de Molécula
3.
Nat Genet ; 50(6): 814-824, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29808028

RESUMO

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.

4.
Cell ; 171(7): 1559-1572.e20, 2017 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-29245011

RESUMO

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.


Assuntos
Modelos Animais de Doenças , Melanoma/metabolismo , RNA Longo não Codificante/metabolismo , Peixe-Zebra , Animais , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Proteínas de Ligação a RNA/metabolismo , Testículo/metabolismo
5.
Nat Cell Biol ; 19(12): 1400-1411, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29180822

RESUMO

The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11-RAD50-NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA-RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.


Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , RNA Longo não Codificante/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Sistema Livre de Células , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteína Homóloga a MRE11/metabolismo , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/genética , RNA Polimerase II/metabolismo , RNA Longo não Codificante/genética , Transcrição Genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
7.
Cell Rep ; 19(3): 630-642, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28423324

RESUMO

Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecular-weight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs.


Assuntos
MicroRNAs/metabolismo , Imagem Individual de Molécula/métodos , Animais , Proteínas Argonauta/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Espaço Intracelular/metabolismo , Camundongos , MicroRNAs/genética , Modelos Biológicos , Sondas Moleculares/metabolismo , Estabilidade de RNA , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Frações Subcelulares/metabolismo
9.
Cancer Cell ; 31(4): 532-548.e7, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28344039

RESUMO

Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides (EIPs) and derived peptidomimetics bound ERG with high affinity and specificity, leading to proteolytic degradation of the ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion and proliferation, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other malignancies.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Peptidomiméticos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Embrião de Galinha , DNA/metabolismo , Humanos , Masculino , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas de Fusão Oncogênica/genética , Biblioteca de Peptídeos , Peptidomiméticos/química , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Domínios Proteicos , Regulador Transcricional ERG/antagonistas & inibidores , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Commun ; 8: 13980, 2017 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-28239143

RESUMO

The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.


Assuntos
Dano ao DNA , RNA/metabolismo , Telômero/metabolismo , Animais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/farmacologia , RNA/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , Ribonuclease III/metabolismo , Transcrição Genética/efeitos dos fármacos
11.
Nat Commun ; 7: 12791, 2016 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-27666543

RESUMO

Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise <1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1. We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , RNA Longo não Codificante/metabolismo , Antineoplásicos Hormonais/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , RNA Longo não Codificante/genética , Receptores Estrogênicos , Tamoxifeno/farmacologia
12.
Cell Rep ; 14(6): 1448-1461, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26854235

RESUMO

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling.


Assuntos
Proteínas Argonauta/genética , Transformação Celular Neoplásica/genética , Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Proteínas Argonauta/antagonistas & inibidores , Proteínas Argonauta/metabolismo , Sequência de Bases , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Inativação Gênica , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transgenes
13.
Sci Signal ; 8(358): ra1, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25564677

RESUMO

Transforming growth factor-ß (TGF-ß) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-ß, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-ß signaling. BUB1 interacted with TGFBRI in the presence of TGF-ß and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-ß-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-ß signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-ß signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-ß signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-ß pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Proteínas Smad Reguladas por Receptor/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Animais , Western Blotting , Linhagem Celular Tumoral , Dimerização , Imunofluorescência , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Fatores de Crescimento Transformadores beta/química , Transdução de Sinais/genética
14.
Biopolymers ; 103(5): 296-302, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25546606

RESUMO

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.


Assuntos
Microscopia de Fluorescência , Congressos como Assunto
16.
Nat Commun ; 4: 2414, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24008311

RESUMO

The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional (3D) pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms, interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the centre of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.


Assuntos
Imagem Tridimensional/métodos , Poro Nuclear/metabolismo , Transporte de RNA , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Difusão , Células HeLa , Humanos , Microscopia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo
17.
Methods ; 63(2): 188-99, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23820309

RESUMO

Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action.


Assuntos
MicroRNAs/metabolismo , Análise de Célula Única/métodos , Animais , Sequência de Bases , Corantes Fluorescentes/química , Genes Reporter , Células HeLa , Humanos , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , MicroRNAs/química , MicroRNAs/genética , Microinjeções , Microscopia de Fluorescência , Interferência de RNA , Ribonucleoproteínas/metabolismo
18.
EMBO Rep ; 13(8): 709-15, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22688967

RESUMO

MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.


Assuntos
Espaço Intracelular/metabolismo , MicroRNAs/metabolismo , Microscopia/métodos , Transdução de Sinais/genética , Animais , Difusão , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Cinética , Camundongos , Microinjeções , Modelos Biológicos , Fotodegradação , Transporte de RNA/genética , Fatores de Tempo
19.
J Biol Chem ; 284(39): 26732-41, 2009 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-19542234

RESUMO

Despite extensive characterization of the mu-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A mu-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [(35)S]guanosine 5'-(gamma-thio)triphosphate binding to Galpha(i), and is allosterically regulated by coupled G(i) protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys(7), Cys(8)]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the mu-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.


Assuntos
Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Receptores Opioides mu/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Linhagem Celular , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Peptídeos Opioides/metabolismo , Peptídeos Opioides/farmacologia , Ligação Proteica , Receptores Opioides mu/genética , Receptores Opioides mu/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera
20.
Chem Soc Rev ; 35(11): 1111-21, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17057840

RESUMO

For the most part DNA was considered Nature's instruction manual for life leading to the popular description 'blueprint of life'. However, DNA is now taking on a new aspect where it is finding use as a construction element for architecture on the nanoscale. This tutorial review addresses the importance of building ordered structures with DNA on the nanoscale, the underlying principles and approaches to build such scaffolds, the current limitations and the anticipated trajectory of the area. This is would be of interest to the chemical biology, supramolecular and bioengineering communities in particular.


Assuntos
DNA/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Conformação de Ácido Nucleico
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