The use of newly discovered inhibitors to identify novel components of the ER associated degradation pathway.

Living things are made up of one or more cells, and cells need membranes to provide barriers inside the cell and to protect them from the outside world. Whilst the biological membranes that form these barriers are incredibly complex and diverse in nature, they are generally characterised by a lipid bilayer studded with many proteins. In order to make new membranes, the cell has to put new proteins into the lipid bilayer and in higher cells this is one of the key functions of a specialised compartment known as the endoplasmic reticulum. The endoplasmic reticulum contains a number of molecular machines that stitch new proteins into the lipid bilayer to make functional membranes. It has become increasing clear over recent years that sometime this process goes wrong, for example because of a mutation or defect in the protein being made. In order to sort such bad proteins from the good ones, the endoplasmic reticulum also has a set of molecular machines that check all the proteins it has made, only the good proteins are used and the defective ones are destroyed. This so called 'quality control' process is of particular interest because it linked to some kinds of disease, for example cystic fibrosis, and it can also restrict the production of proteins for biotechnology applications. The aim of this project is to use a newly discovered drug to block the destruction of two different kinds of defective proteins allowing us to identify the cellular machines that recognise them. This will help us understand how the cell knows the difference between good proteins and bad proteins, and in the longer term may help us to manipulate this process to our benefit.

The principal goal of this project is to identify novel cellular components responsible for the selection and delivery of aberrant membrane proteins to the pathway for endoplasmic reticulum associated degradation (ERAD) via the process of quality control. Whilst the later stages of the ERAD pathway are relatively well defined, it is clear that there are a number of distinct routes that feed into this common downstream element of the process. These distinct early stages represent the action of multiple 'chaperone-like' components that recognise different features of aberrant proteins synthesised at the ER and target them for destruction via the ERAD pathway. We have chosen two model substrates that are delivered via novel, and as yet undefined routes to the ERAD pathway. During the course of this project we will identify the cellular factors that specifically associate with these two classes of ERAD substrates and establish which components are directly implicated in their quality control. To achieve this goal, we will use the newly discovered drug eeyarestatin to induce an artificial blockade of these novel ERAD pathways. This approach will allow us to accumulate model ERAD substrates in stable complexes with candidate ERAD facilitators and thereby identify them. Since the use of the drug eeyarestatin is pivotal to this project, we will undertake its de novo synthesis and validation at the outset of the work. This will provide us with direct, and unfettered, access to the amounts of the compound required for the project. Following their identification, candidate components will be further analysed in order to identify bona fide ERAD facilitators. The function of these proteins will then be carefully defined by manipulating them in vivo in do as to determine the basis for their action as ERAD facilitators. Taken together, this project will allow us to identify the cellular components that are responsible for the targeted delivery of novel substrates to the ERAD pathway and enable us to define the role of these components within the framework of ER quality control as a whole.