Developing molecular therapy approaches for cystic fibrosis

Background:
Cystic Fibrosis is a life-shortening genetic condition that results from insufficient activity of the CFTR transporter at the cell surface. Most cases of Cystic Fibrosis are caused by mutations in CFTR that retain the protein in the endoplasmic reticulum (ER). Therapeutic strategies designed to allow mutant CFTR out of the ER may hold the answer. Indeed, promotion of ER-export is sufficient to be therapeutically effective. We have identified functionally and structurally different membrane protein cargoes in yeast that are trapped in the ER but can be efficiently exported under certain conditions. The budding yeast system has unsurpassed genetic tools to aide in the discovery of novel and conserved membrane trafficking machineries, such as the Nobel prize winning dissection of the secretory and autophagy pathways.

Research plan:
Following exit from the ER, cargoes traverse the Golgi apparatus en route to the surface. Although surface recycling can occur through different routes, cargoes eventually accumulate in the yeast lysosome, an easy phenotype to score successful ER export, even using high throughput methods. We have shown conditions that both promote and inhibit ER trafficking, demonstrating the pathway is physiologically regulated and providing an experimental handle to understand the molecular mechanisms governing the process. Initial work is aimed at understanding how certain proteins and metabolic conditions contribute to ER export. Additionally, by screening yeast mutants using robotic protocols, we can identify novel and conserved factors that influence ER-export. Elucidating the molecular mechanisms of proteins involved in ER trafficking in yeast will guide experimentation of the human orthologues in cultured mammalian cells.

Summary:
The PhD candidate will use cell and molecular biology, biochemistry and genetic approaches, combined with sophisticated imaging and cytometry techniques at the Bioscience Technology Facility at York to achieve these goals. Furthermore, training in yeast and mammalian cell culture will expose the student to the advantages of each experimental system. This work aims to identify targets and therapeutic strategies to stimulate trafficking from the ER and promote proteins like mutant CFTR to reach the cell surface. This effort will also provide a framework for small molecule drug screening. Finally, the proposed work complements the current focus of the lab in understanding cell surface recycling, which also regulates CFTR activity.