RESUMO
The nascent polypeptide-associated complex (NAC) is a conserved ribosome-associated protein biogenesis factor. Whether NAC exerts chaperone activity and whether this function is restricted to de novo protein synthesis is unknown. Here, we demonstrate that NAC directly exerts chaperone activity toward structurally diverse model substrates including polyglutamine (PolyQ) proteins, firefly luciferase, and Aß40. Strikingly, we identified the positively charged ribosome-binding domain in the N terminus of the ßNAC subunit (N-ßNAC) as a major chaperone entity of NAC. N-ßNAC by itself suppressed aggregation of PolyQ-expanded proteins in vitro, and the positive charge of this domain was critical for this activity. Moreover, we found that NAC also exerts a ribosome-independent chaperone function in vivo. Consistently, we found that a substantial fraction of NAC is non-ribosomal bound in higher eukaryotes. In sum, NAC is a potent suppressor of aggregation and proteotoxicity of mutant PolyQ-expanded proteins associated with human diseases like Huntington's disease and spinocerebellar ataxias.
Assuntos
Peptídeos beta-Amiloides/genética , Chaperonas Moleculares/genética , Agregação Patológica de Proteínas/genética , Peptídeos beta-Amiloides/química , Sítios de Ligação/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Luciferases/química , Luciferases/genética , Chaperonas Moleculares/química , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Domínios Proteicos/genética , Dobramento de Proteína , Ribossomos/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5'-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.
Assuntos
Processamento Alternativo , Caenorhabditis elegans/genética , Riboswitch , Tetraciclina/farmacologia , Processamento Alternativo/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos , Éxons , Expressão Gênica , Células HeLa , HumanosRESUMO
As newly synthesized polypeptides emerge from the ribosome, it is crucial that they fold correctly. To prevent premature aggregation, nascent chains interact with chaperones that facilitate folding or prevent misfolding until protein synthesis is complete. Nascent polypeptide-associated complex (NAC) is a ribosome-associated chaperone that is important for protein homeostasis. However, how NAC binds its substrates remains unclear. Using native electrospray ionization MS (ESI-MS), limited proteolysis, NMR, and cross-linking, we analyzed the conformational properties of NAC from Caenorhabditis elegans and studied its ability to bind proteins in different conformational states. Our results revealed that NAC adopts an array of compact and expanded conformations and binds weakly to client proteins that are unfolded, folded, or intrinsically disordered, suggestive of broad substrate compatibility. Of note, we found that this weak binding retards aggregation of the intrinsically disordered protein α-synuclein both in vitro and in vivo These findings provide critical insights into the structure and function of NAC. Specifically, they reveal the ability of NAC to exploit its conformational plasticity to bind a repertoire of substrates with unrelated sequences and structures, independently of actively translating ribosomes.
Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/química , Peptídeos/metabolismo , Biossíntese de Proteínas , Sinucleínas/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Chaperonas Moleculares/metabolismo , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Sinucleínas/metabolismoRESUMO
The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Here we describe tetracycline-dependent ribozymes as versatile RNA-based genetic switches in C. elegans. Ribozyme insertion into the 3'-UTR converts any gene of interest into a tetracycline-inducible gene allowing temporal and, by using tissue-selective promoters, spatial control of expression in all developmental stages of the worm. Using the ribozyme switches we established inducible C. elegans polyglutamine Huntington's disease models exhibiting ligand-controlled polyQ-huntingtin expression, inclusion body formation, and toxicity. Our approach circumvents the complicated expression of regulatory proteins. Moreover, only little coding space is necessary and natural promoters can be utilized. With these advantages tetracycline-dependent ribozymes significantly expand the genetic toolbox for C. elegans.