RESUMO
Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear. Here we show that a single historical amino acid substitution is able to constrain the subunit stoichiometry of functional acetylcholine receptors. Using a combination of ancestral sequence reconstruction, single-channel electrophysiology, and concatenated subunits, we reveal that an ancestral ß-subunit can not only replace the extant ß-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral ß-subunit with a single amino acid substitution, we restore the requirement for a δ-subunit for functional channels. These findings reveal that a single historical substitution necessitates an increase in acetylcholine receptor complexity and, more generally, that simple stepwise mutations can drive subunit entrenchment in this model heteromeric protein.
Assuntos
Substituição de Aminoácidos , Multimerização Proteica , Receptores Nicotínicos/genética , Linhagem Celular , Evolução Molecular , Humanos , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismoRESUMO
Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties.
Assuntos
Evolução Molecular , Receptores Colinérgicos/química , Animais , Humanos , Estrutura Terciária de Proteína , Receptores Colinérgicos/metabolismo , Relação Estrutura-AtividadeRESUMO
Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the ß-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost(-/-)) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. ß-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 (cyp27B1) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion.
Assuntos
Cálcio/urina , Fatores de Crescimento de Fibroblastos/sangue , Glicoproteínas/metabolismo , Vitamina D/sangue , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Densidade Óssea , Cromatografia Líquida , Feminino , Fator de Crescimento de Fibroblastos 23 , Heterozigoto , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mutação , Osteoblastos/citologia , Osteoclastos/citologia , Osteócitos/citologia , Microtomografia por Raio-X , beta-Galactosidase/metabolismoRESUMO
One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.
The human genome counts over 20,000 genes, which can be turned on and off to create the proteins required for most of life processes. Once produced, proteins need move to specific locations in the cell, where they are able to perform their jobs. Despite striking scientific advances, 90% of human genes are still under-studied; where the proteins they code for go, and what they do remains unknown. Zebrafish share many genes with humans, but they are much easier to manipulate genetically. Here, Ichino et al. used various methods in zebrafish to create a detailed 'catalogue' of previously poorly understood genes, focusing on where the proteins they coded for ended up and the biological processes they were involved with. First, a genetic tool called gene-breaking transposons (GBTs) was used to create over 1,200 strains of genetically altered fish in which a specific protein was both tagged with a luminescent marker and unable to perform its role. Further analysis of 204 of these strains revealed new insight into the role of each protein, with many having unexpected roles and localisations. For example, in one zebrafish strain, the affected gene was similar to a human gene which, when inactivated, causes severe muscle weakness. These fish swam abnormally slowly and also had muscle problems, suggesting that the GBT fish strains could 'model' the human disease. This work sheds new light on the role of many previously poorly understood genes. In the future, similar collections of GBT fish strains could help researchers to study both normal human biology and disease. They could especially be useful in cases where the genes responsible for certain conditions are still difficult to identify.
Assuntos
Técnicas de Silenciamento de Genes , Biblioteca Gênica , Genes Reporter , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Acetylcholine receptors (AChRs) are members of a superfamily of proteins called pentameric ligand-gated ion channels, which are found in almost all forms of life and thus have a rich evolutionary history. Muscle-type AChRs are heteropentameric complexes assembled from four related subunits (α, ß, δ, and É). Here we reconstruct the amino acid sequence of a ß subunit ancestor shared by humans and cartilaginous fishes (i.e., Torpedo). Then, by resurrecting this ancestral ß subunit and co-expressing it with human α, δ, and É subunits, we show that despite 132 substitutions, the ancestral subunit is capable of forming human/ancestral hybrid AChRs. Whole-cell currents demonstrate that the agonist acetylcholine has reduced potency for hybrid receptors, while single-channel recordings reveal that hybrid receptors display reduced conductance and open probability. Our results outline a promising strategy for studies of AChR evolution aimed at identifying the amino acid origins of AChR structure and function.
Assuntos
Proteínas de Peixes/química , Receptores Colinérgicos/química , Homologia de Sequência de Aminoácidos , Acetilcolina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , Evolução Molecular , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Humanos , Ligação Proteica , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismoRESUMO
Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.
Assuntos
Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Alelos , Animais , Análise Custo-Benefício , Primers do DNA , Genótipo , Análise de Sequência de DNARESUMO
The androgen receptor (AR) is required for castration-resistant prostate cancer (CRPC) progression, but the function and disease relevance of AR-bound enhancers remain unclear. Here, we identify a group of AR-regulated enhancer RNAs (e.g., PSA eRNA) that are upregulated in CRPC cells, patient-derived xenografts (PDXs), and patient tissues. PSA eRNA binds to CYCLIN T1, activates P-TEFb, and promotes cis and trans target gene transcription by increasing serine-2 phosphorylation of RNA polymerase II (Pol II-Ser2p). We define an HIV-1 TAR RNA-like (TAR-L) motif in PSA eRNA that is required for CYCLIN T1 binding. Using TALEN-mediated gene editing we further demonstrate that this motif is essential for increased Pol II-Ser2p occupancy levels and CRPC cell growth. We have uncovered a P-TEFb activation mechanism and reveal altered eRNA expression that is related to abnormal AR function and may potentially be a therapeutic target in CRPC.
Assuntos
Fator B de Elongação Transcricional Positiva/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , RNA/metabolismo , Receptores Androgênicos/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Ciclina T/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Humanos , Masculino , Modelos Biológicos , Motivos de Nucleotídeos/genética , Fosforilação , Antígeno Prostático Específico/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Ligação Proteica , RNA Polimerase II/metabolismo , Serina/metabolismo , Regulação para Cima/genéticaRESUMO
Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.
Assuntos
Engenharia Genética/métodos , Genômica/métodos , Efetores Semelhantes a Ativadores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Marcação de Genes , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Cholangiocarcinoma is a highly lethal cancer with limited therapeutic options. Recent genomic analysis of cholangiocarcinoma has revealed the presence of fibroblast growth factor receptor 2 (FGFR2) fusion proteins in up to 13% of intrahepatic cholangiocarcinoma (iCCA). FGFR fusions have been identified as a novel oncogenic and druggable target in a number of cancers. In this study, we established a novel cholangiocarcinoma patient derived xenograft (PDX) mouse model bearing an FGFR2-CCDC6 fusion protein from a metastatic lung nodule of an iCCA patient. Using this PDX model, we confirmed the ability of the FGFR inhibitors, ponatinib, dovitinib and BGJ398, to modulate FGFR signaling, inhibit cell proliferation and induce cell apoptosis in cholangiocarcinoma tumors harboring FGFR2 fusions. In addition, BGJ398 appeared to be superior in potency to ponatinib and dovitinib in this model. Our findings provide a strong rationale for the investigation of FGFR inhibitors, particularly BGJ398, as a therapeutic option for cholangiocarcinoma patients harboring FGFR2 fusions.
Assuntos
Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Proteínas do Citoesqueleto/metabolismo , Fusão Gênica , Neoplasias Pulmonares/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/secundário , Proteínas do Citoesqueleto/genética , Humanos , Imidazóis/farmacologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Piridazinas/farmacologia , Quinolonas/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes--fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a--had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a)-ErbB2/3-AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a-ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.
Assuntos
Nadadeiras de Animais/embriologia , Neurregulinas/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Organogênese/genética , Pele/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Alelos , Nadadeiras de Animais/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Mutagênese Insercional , Neurregulinas/genética , Proteínas Oncogênicas v-erbB/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Pele/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Imagine for a moment being in a small, cold, dark, and dirty room. You haven't seen your family in months and you're not sure if you ever will again. When the drugs that you've given begin to wear off, you feel hunger pangs because you haven't eaten anything in more hours than you can count. You hear a door opening and are filled with paralyzing fear and dread. You are never quite sure who or what will greet you on the other side of that door. You may have to endure a brutal beating, you may be forced to take drugs, or you may be raped.
Assuntos
Tráfico de Pessoas , Vítimas de Crime , Tráfico de Pessoas/legislação & jurisprudência , Tráfico de Pessoas/prevenção & controle , Tráfico de Pessoas/psicologia , Humanos , Papel do Profissional de Enfermagem , Exame Físico , Estados UnidosRESUMO
Sclerostin is a highly conserved, secreted, cystine-knot protein which regulates osteoblast function. Humans with mutations in the sclerostin gene (SOST), manifest increased axial and appendicular skeletal bone density with attendant complications. In adult bone, sclerostin is expressed in osteocytes and osteoblasts. Danio rerio sclerostin-like protein is closely related to sea bass sclerostin, and is related to chicken and mammalian sclerostins. Little is known about the expression of sclerostin in early developing skeletal or extra-skeletal tissues. We assessed sclerostin (sost) gene expression in developing zebrafish (D. rerio) embryos with whole mount is situ hybridization methods. The earliest expression of sost mRNA was noted during 12h post-fertilization (hpf). At 15 hpf, sost mRNA was detected in the developing nervous system and in Kupffer's vesicle. At 18, 20 and 22 hpf, expression in rhombic lip precursors was seen. By 24 hpf, expression in the upper and lower rhombic lip and developing spinal cord was noted. Expression in the rhombic lip and spinal cord persisted through 28 hpf and then diminished in intensity through 44 hpf. At 28 hpf, sost expression was noted in developing pharyngeal cartilage; expression in pharyngeal cartilage increased with time. By 48 hpf, sost mRNA was clearly detected in the developing pharyngeal arch cartilage. Sost mRNA was abundantly expressed in the pharyngeal arch cartilage, and in developing pectoral fins, 72, 96 and 120 hpf. Our study is the first detailed analysis of sost gene expression in early metazoan development.
Assuntos
Osso e Ossos/metabolismo , Encéfalo/metabolismo , Cartilagem/metabolismo , Glicoproteínas/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Osso e Ossos/embriologia , Encéfalo/embriologia , Cartilagem/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Transcrição Gênica , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3))].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early highly restricted, and subsequent massive changes in more than 10% of expressed cellular RNA were observed. At days post fertilization (dpf) 2 [24 h 1α,25(OH)(2)D(3)-treatment], only four RNAs were differentially expressed (hormone vs. vehicle). On dpf 4 (72 h treatment), 77 RNAs; on dpf 6 (120 h treatment) 1039 RNAs; and on dpf 7 (144 h treatment), 2407 RNAs were differentially expressed in response to 1α,25(OH)(2)D(3). Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)(2)D(3) vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)(2)D(3)-treated larvae, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL, and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1α,25(OH)(2)D(3) functions in vivo in developing eukaryotes.