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1.
Nature ; 529(7586): 358-363, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26760206

RESUMO

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.


Assuntos
Códon/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Genes Sintéticos/genética , Meia-Vida , Cinética , Modelos Logísticos , Modelos Genéticos , Dados de Sequência Molecular , Razão de Chances , Elongação Traducional da Cadeia Peptídica , Dobramento de RNA , Estabilidade de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Transcrição Gênica/genética , Proteínas Virais/metabolismo
2.
Exp Cell Res ; 319(12): 1759-1773, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23588206

RESUMO

Non-apoptotic cell death mechanisms are largely uncharacterized despite their importance in physiology and disease [1]. Here we sought to systematically identify non-apoptotic cell death pathways in mammalian cells. We screened 69,612 compounds for those that induce non-canonical cell death by counter screening in the presence of inhibitors of apoptosis and necrosis. We further selected compounds that require active protein synthesis for inducing cell death. Using this tiered approach, we identified NID-1 (Novel Inducer of Death-1), a small molecule that induces an active, energy-dependent cell death in diverse mammalian cell lines. NID-1-induced death required components of the autophagic machinery, including ATG5, and the lysosomal hydrolase cathepsin L, but was distinct from classical macroautophagy. Since macroautophagy can prevent cell death in several contexts, we tested and found that NID-1 suppressed cell death in a cell-based model of Huntington's disease, suggesting that NID-1 activates a specific pathway. Thus the discovery of NID-1 identifies a previously unexplored cell death pathway, and modulating this pathway may have therapeutic applications. Furthermore, these findings provide a proof-of-principle for using chemical screening to identify novel cell death paradigms.


Assuntos
Autofagia/efeitos dos fármacos , Catepsina L/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Tiofenos/farmacologia , Animais , Apoptose , Proteína 5 Relacionada à Autofagia , Ensaios de Triagem em Larga Escala , Proteína Huntingtina , Camundongos , Necrose , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Células PC12 , Peptídeos/toxicidade , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia
3.
Nat Chem Biol ; 6(12): 900-6, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21079601

RESUMO

A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction and oxidation of disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aß peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI represents a new mechanism linking protein misfolding and apoptotic cell death.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Deficiências na Proteostase/patologia , Marcadores de Afinidade , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/patologia , Dissulfetos/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Éxons/genética , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Chaperonas Moleculares/fisiologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Células PC12 , Dobramento de Proteína , Ratos , Transdução de Sinais/fisiologia , Bibliotecas de Moléculas Pequenas
5.
ACS Chem Biol ; 8(5): 914-22, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23496866

RESUMO

Small molecule modulators of protein activity have proven invaluable in the study of protein function and regulation. While inhibitors of protein activity are relatively common, small molecules that can increase protein abundance are rare. Small molecule protein upregulators with targeted activities would be of value in the study of the mechanisms underlying loss-of-function diseases. We developed a high-throughput screening approach to identify small molecule upregulators of the Survival of Motor Neuron protein (SMN), whose decreased levels cause the neurodegenerative disease spinal muscular atrophy (SMA). We screened 69,189 compounds for SMN upregulators and performed mechanistic studies on the most active compound, a bromobenzophenone analogue designated cuspin-1. Mechanistic studies of cuspin-1 revealed that increasing Ras signaling upregulates SMN protein abundance via an increase in translation rate. These findings suggest that controlled modulation of the Ras signaling pathway may benefit patients with SMA.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteínas do Complexo SMN/genética , Proteínas do Complexo SMN/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Proteínas ras/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Simulação por Computador , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Atrofia Muscular Espinal/patologia , Biossíntese de Proteínas , Piridinas/química , Piridinas/farmacologia , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Proteínas ras/genética
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