Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Nat Commun ; 14(1): 5581, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37696852

RESUMO

C9ORF72 hexanucleotide repeat expansion is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the accumulation of toxic dipeptide repeat (DPR) proteins like poly-GA, GP and GR, produced by the noncanonical translation of the expanded RNA repeats. However, how different DPRs are synthesized remains elusive. Here, we use single-molecule imaging techniques to directly measure the translation dynamics of different DPRs. Besides initiation, translation elongation rates vary drastically between different frames, with GP slower than GA and GR the slowest. We directly visualize frameshift events using a two-color single-molecule translation assay. The repeat expansion enhances frameshifting, but the overall frequency is low. There is a higher chance of GR-to-GA shift than in the reversed direction. Finally, the ribosome-associated protein quality control (RQC) factors ZNF598 and Pelota modulate the translation dynamics, and the repeat RNA sequence is important for invoking the RQC pathway. This study reveals that multiple translation steps modulate the final DPR production. Understanding repeat RNA translation is critically important to decipher the DPR-mediated pathogenesis and identify potential therapeutic targets in C9ORF72-ALS/FTD.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Demência Frontotemporal/genética , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , RNA/genética , Imagem Individual de Molécula , Dipeptídeos , Proteínas de Transporte
2.
Nat Commun ; 12(1): 4908, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389711

RESUMO

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.


Assuntos
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Íntrons/genética , Biossíntese de Proteínas/genética , RNA/genética , Transporte Ativo do Núcleo Celular/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética
3.
Neuron ; 104(5): 885-898.e8, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31587919

RESUMO

Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells. We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/biossíntese , RNA Helicases DEAD-box/metabolismo , Demência Frontotemporal/metabolismo , Animais , Sistemas CRISPR-Cas , Drosophila , Humanos , Biossíntese de Proteínas/fisiologia , Sequências Repetitivas de Ácido Nucleico
4.
Mol Pharm ; 15(11): 5046-5057, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30226785

RESUMO

Human serum albumin (HSA) fusion protein is a viable and effective approach to target and inhibit essential intracellular pathways. It has previously been shown that an HSA fusion protein containing a p53-reactivating peptide (rHSA-p53i) retains the binding activity to MDM2 and MDMX, resulting in p53 transcription-dependent apoptosis. Here, we demonstrate that rHSA-p53i is able to bind and neutralize anti-apoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. This interaction displaces pro-apoptotic Bak and subsequently leads to intrinsic apoptosis via mimicking a p53 transcription-independent pathway. Cytotoxicity induced by rHSA-p53i, via p53 transcription dependent and independent apoptotic pathways, is irrespective of the p53 status in MDA-MB-231, HeLa, and SJSA-1 cells possessing either mutant, deficient, or wild-type p53. The therapeutic potential is also confirmed by treating SJSA-1 and MDA-MB-231 xenograft mouse tumors with rHSA-p53i. These data reveal that rHSA-p53i interferes with at least four intracellular targets, making it a viable therapeutic protein for the treatment of a variety of cancers, as well as a carrier to deliver fatty acid-modified chemotherapeutics.


Assuntos
Neoplasias/tratamento farmacológico , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica Humana/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Peptídeos/genética , Peptídeos/uso terapêutico , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Albumina Sérica Humana/genética , Albumina Sérica Humana/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Methods Mol Biol ; 1751: 109-125, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29508293

RESUMO

The vital role of microRNAs (miRNAs) involved in gene expression regulation has been confirmed in many biological processes. With the growing power and reducing cost of next-generation sequencing, more and more researchers turn to apply this high-throughput method to solve their biological problems. For miRNAs with known sequences, their expression profiles can be generated from the sequencing data. It also allows us to identify some novel miRNAs and explore the sequence variations under different conditions. Currently, there are a handful of tools available to analyze the miRNA sequencing data with separated or combined features, such as reads preprocessing, mapping and differential expression analysis. However, to our knowledge, a hands-on guideline for miRNA sequencing data analysis covering all steps is not available. Here we will utilize a set of published tools to perform the miRNA analysis with detailed explanation. Particularly, the miRNA target prediction and annotation may provide useful information for further experimental verification.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/análise , MicroRNAs/genética , Análise de Sequência de RNA/métodos , Humanos
6.
Mol Pharm ; 13(10): 3370-3380, 2016 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-27546214

RESUMO

Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (ZHER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of ZHER2:342 has limited its pharmaceutical application. Human serum albumin (HSA) and ZHER2:342 fusion protein may not only extend the serum half-life of ZHER2:342 but also preserve the biological function of HSA to bind and transport fatty acids, which can be used to deliver fatty acid-modified therapeutics to HER2-positive cancer cells. Two HSA and ZHER2:342 fusion proteins, one with a single ZHER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and another with two tandem copies of ZHER2:342 fused to the C terminus of HSA (rHSA-(ZHER2)2), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)2, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)2 treatment has been observed. HSA fusion proteins are easy and economical to express, purify, and formulate. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified FITC could be efficiently taken up by cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.


Assuntos
Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Albumina Sérica/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ácidos Graxos/química , Humanos , Imunoprecipitação , Células MCF-7 , Fosforilação , Ligação Proteica , Receptor ErbB-2/química , Proteínas Recombinantes de Fusão/química , Albumina Sérica/química
7.
Curr Pharm Des ; 21(14): 1899-907, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25732550

RESUMO

Fusion proteins have been well-studied and widely applied in biopharmaceutics. Albumin fusion proteins are simple to construct, easy to purify, and stable to formulate. One main application of fusion protein is to extend the plasma half-life of therapeutic proteins and peptides. Albiglutide for diabetes treatment is the first albumin fusion protein drug approved by FDA. Balugrastim and other albumin fusion proteins have been evaluated in clinical trials. Taking advantage of the physiological functions of albumin, albumin fusion proteins can also been applied to act on an essential intracellular target and carry fatty acid-modified drugs. This novel approach makes it possible to co-deliver two different types of drug to one tumor cell for synergistic cytotoxicity.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteínas Recombinantes de Fusão/administração & dosagem , Albumina Sérica/administração & dosagem , Animais , Sistemas de Liberação de Medicamentos/tendências , Humanos , Proteínas Recombinantes de Fusão/química , Albumina Sérica/química
8.
Hum Mol Genet ; 24(8): 2297-307, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25556185

RESUMO

Functional defects of the mitochondrial translation machinery, as a result of mutations in nuclear-encoded genes, have been associated with combined oxidative phosphorylation (OXPHOS) deficiencies. We report siblings with congenital sensorineural deafness and lactic acidemia in association with combined respiratory chain (RC) deficiencies of complexes I, III and IV observed in fibroblasts and liver. One of the siblings had a more severe phenotype showing progressive hepatic and renal failure. Whole-exome sequencing revealed a homozygous mutation in the gene encoding mitochondrial ribosomal protein S7 (MRPS7), a c.550A>G transition that encodes a substitution of valine for a highly conserved methionine (p.Met184Val) in both affected siblings. MRPS7 is a 12S ribosomal RNA-binding subunit of the small mitochondrial ribosomal subunit, and is required for the assembly of the small ribosomal subunit. Pulse labeling of mitochondrial protein synthesis products revealed impaired mitochondrial protein synthesis in patient fibroblasts. Exogenous expression of wild-type MRPS7 in patient fibroblasts rescued complexes I and IV activities, demonstrating the deleterious effect of the mutation on RC function. Moreover, reduced 12S rRNA transcript levels observed in the patient's fibroblasts were also restored to normal levels by exogenous expression of wild-type MRPS7. Our data demonstrate the pathogenicity of the identified MRPS7 mutation as a novel cause of mitochondrial RC dysfunction, congenital sensorineural deafness and progressive hepatic and renal failure.


Assuntos
Acidose Láctica/genética , Perda Auditiva Neurossensorial/genética , Falência Hepática/genética , Proteínas Mitocondriais/genética , Insuficiência Renal/genética , Proteínas Ribossômicas/genética , Acidose Láctica/metabolismo , Adolescente , Sequência de Bases , Criança , Pré-Escolar , Progressão da Doença , Feminino , Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/metabolismo , Humanos , Lactente , Falência Hepática/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas , Insuficiência Renal/metabolismo , Proteínas Ribossômicas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA