AUTO-UBIQUITYLATION

Structural insight into auto-ubiquitylation as a regulatory mechanism for protein trafficking

 Partecipanti

Mappa

 Word cloud

Esplora la "nuvola delle parole (Word Cloud) per avere un'idea di massima del progetto.

 Obiettivo del progetto (Objective)

'The cell interprets the information conferred by ubiquitin (Ub) through Ub-receptors, proteins that harbor ubiquitin-binding domains (UBDs). Ub-receptors can be found in three mutually exclusive forms. Apo is the unbound form, whereas trans is the intermolecular form bound to ubiquitylated cargo. Intriguingly, the third form, cis, is of Ub-receptors that are themselves ubiquitylated. Monoubiquitylation of the Ub-receptors imposes an auto-inhibitory conformation, rendering them unable to bind ubiquitylated cargos, and thus providing an intrinsic regulatory mechanism. While several structures of apo and trans are available, a cis structure form of an ubiquitylated Ub-receptor has yet to be determined. Determining a cis structure is crucial for understanding the molecular mechanism of auto-inhibition. Due to the action of deubiquitylating enzymes, the ubiquitylated form of the Ub-receptors is labile and therefore it is difficult to purify them from eukaryotes. To fill this gap in knowledge, we developed a novel bacterial system for protein ubiquitylation. The system produces a high yield of ubiquitylated protein that can be easily purified and subjected to crystallization trials. Using this bacterial ubiquitylation system, we will determine cis as well as apo and trans structures of Ub-receptors. Guided by comparison of the structures, point mutations will be introduced into key residues. In vitro functional studies including the determination of oligomeric stages, and affinity measurements using isothermal titration calorimetry and analytical ultracentrifugation will be performed on all three forms. Finally, the phenotype of the wild type and mutant proteins will be studied in vivo by monitoring trafficking of GFP-cargo under confocal microscopy. The outcome of this study is predicted to provide the molecular principles of ub-receptor regulation, and serve to design drugs for diseases that involve the monoubiquitin pathways such as AIDS and cancer.'

Introduzione (Teaser)

Proteins use a special sign language when interacting within a cell. One key signal is a small protein called ubiquitin (Ub).

Descrizione progetto (Article)

Abnormalities in the ubiquitin system are implicated in a wide range of pathologies, including certain cancers. Examples are breast and colon cancer, Parkinson's disease, Alzheimer's disease, diabetes and even infectious diseases such as HIV and Ebola. More knowledge about the way proteins signal and how cells decode these signals could unlock the path to new treatments for these major diseases.

The EU-funded 'Auto-ubiquitylation project is investigating how decoding mechanisms function at the molecular' (cellular) level. This is important because Ub-receptors are known to decode the signals for thousands of ubiquitylated proteins.

A detailed study of how Ub-receptors function reveals much about their role in disease (prevention). Research is being done with biochemical and biophysical assays to investigate whether open and closed structures are involved in the function of Ub-receptors.

What the project proposes is that Ub-receptors can 'acquire' three structural states: 'apo', which is the unbound form, 'trans' which is bound to another ubiquitylated-protein, and 'cis' which is self-ubiquitylated. Determining a cis structure has been crucial for understanding how molecules interact among themselves to regulate protein function (auto-inhibition). So the project developed a novel bacterial system for protein ubiquitylation that will help determine high-resolution structures of Ub-receptors in cis form and give better understanding of how auto-regulation takes place.

Project partners expect that their work will make it possible to pharmacologically manipulate the Ub system. Results in this area will serve as templates for the future design of new drugs for diseases such as AIDS and cancer.