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1.
Protein Sci ; 33(4): e4940, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38511482

RESUMO

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.


Assuntos
Estradiol , Receptor alfa de Estrogênio , Animais , Camundongos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Estradiol/química , Estradiol/metabolismo , Ligantes , Tamoxifeno/farmacologia , Tamoxifeno/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mamíferos
2.
Nucleic Acids Res ; 48(22): 12983-12999, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33270897

RESUMO

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.


Assuntos
Trifosfato de Adenosina/análogos & derivados , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Proteínas Virais/genética , Trifosfato de Adenosina/genética , Microscopia Crioeletrônica , DNA Helicases/genética , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Dependovirus/ultraestrutura , Humanos , Proteínas Virais/ultraestrutura
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