Role of the nascent polypeptide-associated complex NAC in proteostasis during aging and disease conditions

Many age-related neurodegenerative diseases such as Alzheimer's, Parkinson's and polyglutamine (PolyQ) diseases, including Huntington's disease, are associated with the aberrant accumulation of specific protein aggregates in affected nerve cells. The accumulation of these protein aggregates, which reflects an age-related failure of the cell's protein quality control system, is causally linked to the pathogenesis of such protein conformational disorders, also called proteinopathies. To maintain protein homeostasis (proteostasis), cells invest in a large network of factors that counteract the misfolding and aggregation of proteins. At the top of this proteostasis network are molecular chaperones that specifically bind and protect unstructured polypeptides from aggregation. Some of these chaperone systems act directly at the ribosome tunnel exit and support the folding, maturation and transport of newly synthesized polypeptide chains in a co-translational manner. Although it is known that ribosome-associated chaperones have a crucial impact on the overall cellular proteostasis, their potential direct effect on disease-related protein aggregation has not yet been investigated. This dissertation focuses on the molecular chaperone function of one of these ribosome-associated factors, the eukaryotic nascent polypeptide-associated complex (NAC), and its role in preventing the accumulation of pathogenic protein aggregatespublishe

Ordnung am Ribosom : der multifunktionale Komplex NAC

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A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans

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.publishe

Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans

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.publishe

Data associated with 'Conformational flexibility within the nascent polypeptide–associated complex enables its interactions with structurally diverse client proteins'

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 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 analysed 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 having unrelated sequences and structures independently of actively translating ribosome