Directed molecular evolution to generate specific disaggregating chaperones to solubilize toxic PolyQ aggregates

• Forschungsstelle: COST
• Projektnummer: C15.0042
• Projekttitel Englisch: Directed molecular evolution to generate specific disaggregating chaperones to solubilize toxic PolyQ aggregates

Schlüsselwörter (Englisch)

PolyQ aggregates; chaperones; ClpX; directed protein evolution

Forschungsprogramme (Englisch)

COST-Action BM1307 - European network to integrate research on intracellular proteolysis pathways in health and disease

Kurzbeschreibung (Englisch)

In Huntington’s disease (HD), the huntingtin polypeptide contains an abnormally long tract of poly glutamines (polyQ), which is highly prone to aggregation. PolyQ aggregates cause neuro-inflammation and loss of neural tissues, leading to chorea, dementia and death. Cells have evolved proteases that are gated by disaggregating chaperones that can recognize a large array of misfolded protein conformers and unfold them into harmless protease-degradable polypeptides. Yet, in aging humans suffering from HD with abnormally long PolyQ tracts, both lines of cellular defenses remain rather ineffective for unclear reasons, which new mutant disaggregases may help clarify. Aims: To use synthetic biology and directed molecular evolution to generate new disaggregating chaperones that can solubilize toxic PolyQ aggregates and convert them into harmless polypeptides. Experimental approach: We shall perform a gain-of-disaggregation function screen in bacteria, using a plasmid encoding for chloramphenicol acetyl transferase (CAT), fused to a long aggregation-prone polyQ tract that renders CAT inactive by aggregation in the E. coli. This plasmid will co-express a model AAA+ type of disaggregating-unfolding chaperone machinery, ClpX, whose N-terminal domain will be randomly mutagenized. Clones will be selected under increasing chloramphenicol concentrations, in iterative rounds of mutagenesis and screening. We expect to isolate new chaperones effective against PoliQ deseases.

Weitere Hinweise und Angaben (Englisch)

Full name of research-institution/enterprise: Université de Lausanne Département de biologie moléculaire végétale Bâtiment de biologie, room 5427

Partner und Internationale Organisationen (Englisch)

AT; BE; HR; CZ; DK; EE; FI; FR; DE; EL; HU; IS; IE; IL; IT; NL; NO; PL; PT; RO; RS; SI; ES; SE; TR; UK; UA

Abstract (Englisch)

See short description

Datenbankreferenzen (Englisch)

Swiss Database: COST-DB of the State Secretariat for Education and Research Hallwylstrasse 4 CH-3003 Berne, Switzerland Tel. [phone] Swiss Project-Number: C15.0042