Huntingtin Polyglutamine-Dependent Protein Aggregation in Reconstituted Cells

One of the aims of synthetic biology is bottom-up construction of reconstituted human cells for medical uses. To that end, we generated giant unilamellar vesicles (GUVs) that contained a HeLa cell extract, which comprises a cell-free protein synthesis (CFPS) system. Then we expressed Huntingtin protein fragments that contained polyglutamine (polyQ) sequences (Htt-polyQ), a hallmark of Huntington's disease. That system produced polyQdependent protein aggregates, as previously demonstrated in living cells. We next simplified the system by generating GUVs that contained purified human factors, which reconstituted a CFPS system. Htt-polyQ fragments expressed in these GUVs also formed protein aggregates. Moreover, an N-terminal deletion mutant, which had failed to form protein aggregates in living cells, also failed to form protein aggregates in the reconstituted GUVs. Thus, the GUV systems that encapsulated a human CFPS system could serve as reconstituted cells for studying neurological diseases.