Year: 2024
Type: conference-paper
Abstract: Background The main cause of Huntington disease (HD) a CAG triplet expansion in the huntingtin (Htt) gene. This encodes a tandem of glutamines which makes the product of this gene prone to misfold and aggregate, eventually forming inclusion bodies where other proteins can be sequestered. We know that Htt protein is essential for life, but also that reducing its concentration in adults does not compromise viability of mammals. Therefore, partially reducing the level of mHtt, is an interesting approach to find a therapy for HD. In this regard, manipulation of the ubiquitin-proteasome pathway to achieve the degradation of specific targets may be used. Ubiquitin is a peptide added catalytically by E3 ubiquitin ligases, which mark specific targets for degradation. As a novel tool we have combined E3 ligase RING domain with camelid-nanobodies to produce ubiquibodies which can mark specific targets for degradation. Aim Proof-of-concept to set up a E3-ligase ubiquibody assay to reduce the expression of mHtt. Methods and Results To this end, we have isolated ten Htt-specific nanobodies. We have generated a HD model in HeLa cells expressing the caspase-6-fragment of mHtt in frame with mNeonGreen. This allows monitoring of aggregation dynamics of mHtt in vitro. We selected one nanobody and proved its specific binding to Htt in vitro. This protein should find, bind and ubiquitinate wildtype Htt and mHtt, hence sending them to degradation. Subsequently, we are testing the nanobody-E3 ligase constructs in these cells and then apply the procedure to a C. elegans model of HD.
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4402374334