Deciphering co-translational protein folding, assembly and quality control pathways, in health and disease

Descrizione del progetto

L’importanza delle proteine ripiegate correttamente

I meccanismi che garantiscono il corretto ripiegamento e assemblaggio delle proteine nel citoplasma, così «affollato», non sono ben noti, ma il numero crescente di malattie da errato ripiegamento sottolinea l’importanza di questo processo. Il ribosoma emerge come un centro nevralgico all’intersezione cruciale fra sintesi e ripiegamento. Recentemente è stato scoperto che anche la fase finale del ripiegamento, ossia l’assemblaggio in complessi, è coordinata con la traslazione. I membri del progetto RiboFriend, finanziato dall’UE, hanno elaborato un approccio di profilazione del ribosoma per catturare gli eventi co-traslazionali in vivo, anche se non sono ancora stati chiariti i meccanismi che regolano i percorsi di assemblaggio co-traslazionale. Il progetto spiegherà i meccanismi di ripiegamento e degradazione delle proteine co- traslazionali in stato di salute e di malattia, facendo progredire la comprensione delle malattie conformazionali e del processo di invecchiamento e aprendo nuove strade per le terapie.

Obiettivo

The majority of cellular proteins do not function alone; rather acting together to achieve concerted functions. Despite the prevalence of protein complexes, little is known of the mechanisms that ensure their correct folding and assembly in the crowded cytoplasm. The importance of the folding challenge is underscored by the growing number of misfolding diseases, often characterized by aggregation of lonely, unassembled protein-subunits.
At the critical intersection of synthesis and folding, the ribosome is emerging as a hub, guiding the polypeptide-chain interactions with targeting factors, modifying enzymes and folding chaperones. We have recently discovered that even the final step of folding, the assembly into complexes, is coordinated with translation. To capture co-translational events, in vivo, we developed a ribosome profiling approach. This approach revealed that emerging polypeptide-chains are constantly engaged by their partner subunits, protecting them from misfolding (Shiber et al., Nature 2018). However the mechanisms regulating co-translational assembly pathways remain largely obscure. In this proposal, we aim to elucidate co-translational protein folding and degradation mechanisms, in health and disease.
We will: (i) Identify and characterize novel co-translational degradation pathways, by targeting ribosomes synthesizing misfolding-prone subunits. (ii) Elucidate the conformational, energetic and kinetic parameters directing folding and assembly, at the atomic level, by molecular dynamics (iii) Develop RiboFriend a single-molecule in vivo approach to elucidate the interplay of chaperones, degradation and assembly factors, during synthesis. Our collective preliminary results strongly support the feasibility of this proposal. The ability to capture co-translational folding and misfolding pathways, in single-molecule resolution can revolutionize our understanding of conformational diseases and the aging process, opening new horizons for therapy.