RESUMO
An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe small molecule-assisted shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, resulting in the production of an untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized copies of the protein. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto an RNA virus for which no licensed inhibitors exist. As SMASh does not require the permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh involves only a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Isoquinolinas/farmacologia , Proteínas Luminescentes/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Transporte/genética , Chlorocebus aethiops , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/virologia , Cultura Primária de Células , Ligação Proteica , Proteólise , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Células Vero , Proteínas não Estruturais Virais/genéticaRESUMO
Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.
Assuntos
Dependovirus/genética , Marcação de Genes , Engenharia Genética , Células-Tronco Pluripotentes , Humanos , Hipoxantina Fosforribosiltransferase/genéticaRESUMO
A simple, quantitative assay for measuring the oncogenic potential of integrating vectors is needed in order to improve vector design and safety. In this study, we have developed a transient plasmid-based assay to measure the activation of a reporter gene by an adjacent vector provirus. Plasmid pACT contains a luciferase cassette driven by a minimal, enhancerless promoter, into which vector proviruses are inserted upstream for evaluation by luciferase assays and northern blots. In a comparison of analogous vectors based on murine leukemia virus (MLV), human immunodeficiency virus (HIV), and foamy virus (FV), we observed significant enhancer activity and read-through transcription from MLV proviruses, and significant read-through transcription from HIV proviruses. HIV and FV proviruses containing an internal MLV long-terminal repeat (LTR) promoter also had significant enhancer activity, which was not observed with an internal promoter from the murine phosphoglycerate kinase-1 gene, PGK. These results demonstrate that neighboring gene activation can be limited by using internal promoter(s) lacking enhancer activity, especially when present in an FV vector backbone that prevents read-through transcription. Although the pACT assay does not measure oncogenesis directly, it should be useful for screening vectors before more time-consuming and costly animal studies are undertaken.
Assuntos
Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Northern Blotting , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , HIV/genética , Humanos , Células K562 , Vírus da Leucemia Murina/genética , Luciferases/genética , Luciferases/metabolismo , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus Espumoso dos Símios/genética , Ativação Transcricional , TransfecçãoRESUMO
An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9-mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.
Assuntos
Apoptose/genética , Bioengenharia/métodos , Neoplasias/genética , Neoplasias/terapia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Adenoviridae , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Endopeptidases/genética , Humanos , Modelos Teóricos , Neoplasias/patologia , Estabilidade Proteica , Proteólise , Receptor ErbB-2/metabolismo , Transdução de Sinais , Biologia Sintética , Transcrição Gênica , Proteínas não Estruturais Virais/genéticaRESUMO
The phosphoprotein (P) of human parainfluenza virus type 3 (HPIV 3) plays a central role in the viral genome RNA transcription and replication. It acts as an essential cofactor of the RNA polymerase (L) by forming a functional L-P complex, binds to the genomic N-RNA template to recruit the L-P complex for RNA synthesis, and interacts with the nucleocapsid protein (N) to form the encapsidation complex (N-P). We have earlier demonstrated that the P protein forms oligomers (B. P. De, M. A. Hoffman, S. Choudhary, C. C. Huntley, and A. K. Banerjee, 2000, J. Virol. 74, 5886-5895) and in this article we identified the putative oligomerization domain of the P protein and studied the role of this domain in transcription. By computer analyses, we have localized a high-score coiled-coil motif characteristic of oligomerization domain residing between the amino acid residues 423 and 457 of the P protein. Deletion of 12 amino acid residues within this coiled-coil motif (P Delta 439-450) completely abrogated oligomerization, whereas deletion in other regions outside the motif had no significant effect. The mutant P Delta 439-450 was both defective in mRNA synthesis in vitro and minigenome transcription in vivo. Interestingly, the mutant interacted with L to form L-P complex, albeit less efficiently, while its interaction with N protein to form N-P complex and with N-RNA template was similar to the wt P protein. Our results indicate that oligomerization provides a key function to the P protein in the transcription of HPIV 3 genome RNA.