1,721,034 research outputs found
Evaluation of LV insulating systems in inverter FED electric motors by means of partial discharges
No full text
Targeting intracellular Aß with oligomeric conformation sensitive recombinant intrabodies
No full textThe intracellular antibody capture technology: towards the high-throughput selection of functional intracellular antibodies for target validation
No full textSeveral approaches have been developed over the past decade to study the complex interactions that occur in biological system. The ability to carry out a comprehensive genetic analysis of an organism becomes more limited and difficult as the complexity of the organism increases because complex organisms are likely to have not only more genes than simple organisms but also more elaborate networks of interactions among those genes. The development of technologies to systematically disrupt protein networks at the genomic scale would greatly accelerate the comprehensive understanding of the cell as molecular machinery. Intracellular antibodies (intrabodies) can be targeted to different intracellular compartments to specifically interfere with function of selected intracellular gene products in mammalian cells. This technique should prove important for studies of mammalian cells, where genetic approaches are more difficult. In the context of large-scale protein interaction mapping projects, intracellular antibodies (ICAbs) promise to be an important tool to knocking out protein function inside the cell. In this context, however, the need for speed and high throughput requires the development of simple and robust methods to derive antibodies which function within cells, without the need for optimization of each individual ICAb. The successful inhibition of biological processes by intrabodies has been demonstrated in a number of different cells. The performance of antibodies that are intracellularly expressed is, however, somewhat unpredictable, because the reducing environment of the cell cytoplasm in which they are forced to work prevents some antibodies, but not others, to fold properly. For this reason, we have developed an in vivo selection procedure named Intracellular Antibody Capture Technology (IACT) that allows the isolation of functional intrabodies. The IAC technology has been used for the rapid identification of antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by the selected intracellular antibodies. Several optimizations of the IAC technology for protein knock-out have been developed so far. This system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in different cell lines
In vivo selection of intrabodies specifically targeting protein-protein interactions: a general platform for an “undruggable” class of disease targets
No full textProtein-protein interactions represent a major potential drug target for many human diseases, but these are unanimously considered undruggable with small chemical molecules. We have developed 3-SPLINT, a novel technology for the selection of antibodies that are intrinsically endowed with the ability to interfere with a given protein-protein interaction. The selection procedure exploits the recently described yeast SPLINT libraries of intrabodies, adapting them to a reverse-hybrid system, yielding the selection of recombinant antibodies that are able to disrupt a target protein-protein interaction in vivo. This class of antibodies should therefore perturb an individual protein-protein interaction, without perturbing the scaffolding function of the target protein in that complex, or other protein interactions of that same protein. We provide here a proof of concept of the technology, by the de novo selection of antibodies against two distinct interacting protein pairs: the GABARAP, which interact with the gamma2 subunit of GABA(A) receptor, and the p65 protein dimer, involved in the NF-kappaB-mediated signalling transduction pathway. Intrabodies selected against the latter were functionally validated in cells. Such antibodies, by interfering with the dimerization domain of p65, lead to an activation of the NF-kappaB-mediated transcriptional activity, which is normally inhibited by p65 knock-down RNAi. This provides a clear-cut demonstration that interfering with a protein interaction can be functionally very different from physically removing one of the interacting proteins. The 3-SPLINT approach provides a general and finer tool for the functional validation of selected protein interactions in protein networks, and is ideally applied to protein "hubs", displaying multiple distinct interactions. 3-SPLINT will therefore complement RNAi-based approaches, in the toolkit of target validation strategies, and is amenable to the systematic isolation of comprehensive sets of antibodies against most protein-protein interactions of a given protein network
- …
