Ubiquitination as a transient modification in regulated protein trafficking

Proteins are moved from one part of the cell to another by 'protein trafficking'. This is achieved through recognition of information contained within, or attached to, the cargo protein by molecular machinery that specifies delivery from one location to another. The function of many cells in the body require proteins to change location under different circumstances, e.g. in response to an external signal, or during different stages of development. This is achieved through regulated protein trafficking, a process central to many physiological processes including hormone secretion, antigen presentation, cell division and whole body glucose homeostasis. Protein trafficking defects have been identified in many disease states where these processes are perturbed. These include cancer, autoimmune diseases and diabetes. Understanding regulated protein traffic will therefore better arm us against these diseases.

In many examples of regulated trafficking, the signals that specify which trafficking step a protein is sorted into are often added to the protein in response to an external cue and can then be rapidly removed when no longer required. One example of this is the addition of the small protein ubiquitin to other proteins. The addition of ubiquitin (ubiquitination) has long been known to target proteins for degradation by delivering them into a pathway which leads to the lysosome - a compartment within the cell which functions to degrade proteins. E.g. many cell surface receptors that transmit information from outside to inside the cell are ubiquitinated following stimulation. This leads to their internalization and lysosomal delivery and serves to attenuate signaling events. The activity of de-ubiquitinating enzymes (DUBs) that remove ubiquitin has been shown to rescue some receptors from this pathway allowing recycling back to the cell surface, allowing the cell to fine-tune signaling responses. As well as signaling delivery of proteins from the cell surface to the lysosome, ubiquitin can also direct newly synthesized proteins there from the biosynthetic pathway.

This proposal is directed towards understanding the role of ubiquitin in regulated protein traffic, using sorting of the glucose transporter GLUT4 as a paradigm. Insulin stimulates glucose transport into fat and muscle cells by promoting delivery of a glucose transport protein (GLUT4) from a specialized internal store to the cell surface. In this way insulin increases the rate of glucose transport into fat and muscle, and thus controls whole body glucose homeostasis. Insulin-dependent delivery of GLUT4 to the cell surface is an example of regulated membrane traffic and is impaired in the disease states of insulin-resistance and Type-2 diabetes. We have recently demonstrated a role for ubiquitin in the delivery of GLUT4 into its specialized internal store. We have also identified a role for a DUB in GLUT4 traffic. We now aim to test our working model that describes how ubiqutination of GLUT4 sorts it into a lysosomal trafficking pathway. We propose that deubiquitination of GLUT4 then rescues the transporter from lysosomal degradation and that this deubiquitination is critical for sorting GLUT4 into the store from where it is mobilized to the cell surface in response to insulin.

Our working model represents the first example of a DUB rescuing a ubiquitinated protein from lysosomal delivery via the biosynthetic pathway and thus introduces a new paradigm into the field. This study also has the potential to serve as a model for other regulated trafficking systems such as those mentioned above, whose dysfunction can lead to disease states and hence our work may lead to therapeutic interventions for a wide range of diseases including cancer and diabetes. In addition, many parasites and viruses hijack the host cell's trafficking machinery and therefore understanding this process may arm us in battle against such infectious agents.

This project aims to further our knowledge of the role that ubiquitin plays as a trafficking signal through the endosomal system. Ubiquitination is a post translational modification that can alter a protein's fate. Ubiquitination targets proteins to the degradative lysosome, both from the cell surface and from the trans Golgi network (TGN). This pathway serves to attenuate many signalling pathways by down-regulating ubiquitinated cell surface receptors. In some cases, deubiquitinating enzymes (DUBs) serve to fine-tune this down regulation by rescuing some receptors from this fate allowing them to recycle to the plasma membrane.

We have recently demonstrated that ubiquitination is required for the regulated trafficking of the facilitative glucose transporter GLUT4. Ubiquitination does not result in GLUT4 being delivered to the lysosome, but rather its sequestration away from the general endosomal system, in a specialised store, from where it is delivered to the cell surface in response to an insulin signal. We now aim to build on this study, our unpublished data, and work from others to test a novel hypothesis describing a cycle of ubiquitination/deubiquitination that serves to divert GLUT4 from the TGN to lysosome pathway into its specialised store. This work will not only further our understanding of GLUT4 traffic and ubiquitin as a sorting signal, but will also serve as a paradigm for other regulated trafficking pathways that underpin many physiological processes, such as those involved in hormone secretion, neurotransmission, antigen presentation and cell division.
This study will be performed in the model 3T3-L1 adipocyte cell line using widely used cell biology approaches and will pursue 3 main objectives.

1. Characterisation of the ubiquitin-dependent trafficking step of GLUT4.
2. Characterisation of the role of the deubiquitinating enzyme USP25 in GLUT4 traffic.
3. Identification of the ubiquitin ligase responsible for ubiquitinating GLUT4.

This work will be beneficial to all members of the cell biology community interested in the mechanisms that underpin membrane protein sorting and trafficking. Our study aims to develop new concepts at the core of ideas emerging within the international cell biology community. It is our contention that the data provided from this study will stimulate thinking about these topics and help facilitate a paradigm shift in approach. Because we have chosen as a model system the sorting of the insulin-responsive glucose transporter GLUT4, the work will also be of potential interest to the pharmaceutical industry which has a substantial interest in Type-2 diabetes and insulin-resistance (disease states that represent significant problems both worldwide and also specifically to the United Kingdom, not only in terms of morbidity and mortality but also from an economic perspective). Although this study does not seek to address directly whether defects in GLUT4-traffic contribute to these disease states, data obtained from this work will contribute to our understanding of GLUT4 traffic and thus potentially pave the way for developing therapeutic interventions. Clearly, this work will also be of interest to those who study aspects of insulin action, diabetes and metabolism as a result.

It is our intention to present findings from this study not only at national and international cell biology meetings, but also at meetings such as the American Diabetes Association Annual meeting, the European Association for the Study of Diabetes and the FASEB Glucose Transporter Biology meeting - all of which are attended by representatives of major pharmaceutical companies.

It is important to note that findings from this study will be relevant to many different biological systems as regulation of membrane traffic in response to external cues is an important, but poorly understood, phenomenon. Hence, the results obtained, the tools generated, and our experimental approaches may prove of wide general interest. To this end, our findings will be made publicly available through presentation at scientific meetings and publication in open-access journals. Presentation at meetings during the progress of this work is important, as it allows discussion of our work during its progression, allowing both feed-back to us from the community to assist in our experimental design and strategy, and also a feed-out mechanism where our data is disseminated prior to publication to others interested in these or related problems.

Furthermore, both PIs have a track record in engaging the general public - for example by presentation at diabetes support groups and through Scottish Executive organised public meetings. Such meetings are also attended by Members of the Scottish Parliament (MSPs) and other policy makers, offering an opportunity for our research to directly influence the nation's health, wealth and culture. While such developments clearly represent a long-term investment, they are nonetheless critical.

It should be noted that the University of Glasgow has an excellent Corporate Communications department dedicated to highlighting research outputs, and a Commercial Explotation Unit to assist with commercialisation of any research output if and when the need arises.