Neurogenin 3: regulation of protein stability.

Diabetes results from loss or inactivity of cells, called islet cells, that produce insulin. One of the ways that we could treat diabetes is by replacing these cells using stem cells that have been instructed to turn into islets. Neurogenin 3 (NGN3) is a protein that drives formation of islets and the amount of NGN3 protein present in very important. We have found that NGN3 protein is unstable and is rapidly destroyed in the cells of the body. This proposal aims to look at the ways this destruction is controlled. When we understand this, we can make altered forms of NGN3 that are resistant to being broken down. In these intial studies, we will inject these stable forms of NGN3 into tadpoles to see if they are, indeed, present at higher levels and are, therefore, more active that the original protein. Tadpoles are excellent for this study as they are large, develop outside the mother and are easy to inject proteins into. Moreover, we already know that tadpoles respond to normal NGN3 so act as a useful system to test for increased NGN3 activity. We will then see if our altered NGN3s are also stable in mammalian cells in culture.
These studies will pave the way for experiments to see if stabilised NGN3 can be added back to stem cells to encourage them to form islets. If we can find a way to re-supply islet cells to diabetic patients, this may be an effective therapy to stabilise blood sugar levels and reduce the severity of symptoms and morbidity resulting from diabetes, one of the greatest public health concerns of the 21st Century.

Diabetes, caused by a loss or inactivity of beta islet cells in the pancreas, is a major cause of illness and mortality worldwide. The basic helix-loop-helix (bHLH) transcription factor Neurogenin 3 (NGN3) drives decision to adopt an endocrine over an exocrine cell fate during development of the pancreas. Indeed, the absolute level of NGN3 is a central regulator of endocrine differentiation, and if levels of NGN3 are elevated at the appropriate time, the number of cells adopting an insulin-expressing fate is dramatically increased. For this reason, manipulation of NGN3 activity is a focus of considerable interest to researchers who are attempting to promote stem cells in culture to adopt an endocrine pancreatic fate, potentially driving differentiation of islets for diabetes therapy. Here we present preliminary data showing the first evidence that the level of active NGN3 is regulated by control of protein stability via the ubiquitin-proteosome system.
Building on unpublished work about the related NGN2 protein and further preliminary findings on the NGN3 protein, the aims of this proposal are three-fold. Firstly firstly we will confirm that NGN3 is degraded by Ubiquitin-mediated proteolysis using Xenopus egg extracts asa biochemical system, alongside studies of NGN3 degradation in mammalian cultured cells. Secondly we will define sites of poly-ubiquitination of NGN3 and explore how this process is regulated by phosphorylation. Thirdly, we will use this information to test the activity of stabilised forms of NGN3 in reporter assays, in cultured cells and in vivo in Xenopus embryos. These studies will allow us not only to define pathways that regulate NGN3 stability, but also to to design forms of NGN3 that are resistant to degradation. In the future, building on work proposed here, we will be able to test the effects of stablising NGN3 on endocrine pancreas formation in in vivo models and stem cells, potentially leading to new therapeutic approaches to treatment of diabetes.