Regulation of ER-nuclear communication by CREB3 transmembrane transcription factors

Our cells carry out a broad range of functions, some of which are common and some distinct for each cell type. As examples, certain cells in our pancreas sense metabolic signals and regulate secretion of insulin to control variations in blood glucose. Different cells in the pancreas play a role in the secretion of enzymes that help breakdown ingested foodstuffs. Cells within developing bone secrete components that build bone matrix, while cells of our blood system produce and secrete antibodies to combat infection. These particular examples reflect a common critical function with which this application is concerned, namely the proper control of secretion and secretory function.

The secretory pathway is controlled by a specialized apparatus, termed the endoplasmic reticulum (ER). This coordinated network of membrane-bound organelles is ultimately responsible for the general and cell-type specific secretion of all the proteins and other components that carry out the diverse functions in the examples above.
Therefore, malfunction of ER pathways is associated with a wide range of important human diseases. Examples include diabetes and lipid disorders, conditions associated with defective bone formation, and diseases associated with malfunction in secretion and inflammation in the intestine such as ulcerative coilitis. Although the links with human disease are well established, many aspects of the detailed mechanisms regulating ER functions and their role in normal processes and disease progression remain poorly understood.

A general feature of cellular function is the process of homeostasis, i.e. the regulation of internal conditions by a system of feedback controls to stabilize the health and proper functioning of the cell in response to changing conditions. This process frequently involves the control of gene expression. A specialized homeostatic pathway occurs in the ER for the control of cholesterol levels in our bodies. In this pathway a transcription factor is physically associated with the ER, where it senses alterations in cholesterol, and as a result is processed and transported to the nucleus to stimulate cholesterol production. Knowledge of this pathway has contributed significantly to understanding cholesterol balance and imbalance in health and disease.

Through our work, we now know there are additional factors, called CREB3 proteins, that are also physically anchored in the ER and control adaptive responses by relaying information back to the nucleus. Recent work has demonstrated that they play vital functions at the ER and are required, for example, in developing bone cells, for normal cartilage formation, and in liver for the control of secreted fatty acid and triglyceride levels. However we have very little mechanistic understanding of how they function.
The principle aims of this proposal are to understand the detailed mechanisms controlling CREB3 activity and how they regulate these distinct downstream pathways. We will address two main objectives; firstly understanding the signalling pathways that control how these factors are retained in the ER and how they are released and transported to the nucleus; secondly, what proteins they interact with to dictate cellular localisation

Overall, elucidation of the mechanisms controlling CREB3 proteins and their transcriptional regulation of adaptive responses will contribute significantly to our understanding of ER biology and important physiological pathways and potentially yield new insight into disease processes of significant burden in the population. Understanding their role and function may also have application e.g., in manipulating the secretory pathway to increase production of desired products.

The CREB3 transcription factors play key roles in physiological pathways whose dysfunction underpin human disease. They have been shown to be required for the control of triglyceride homeostasis in the liver and are essential for normal bone and cartilage formation. They operate by intricate intercompartmental signalling pathways about which we have little understanding. Our objectives concern the elucidation of the mechanisms controlling CREB3 protein function and their regulation of cellular adaptive responses. There are two themes, each involving molecular biology, biochemical and imaging techniques to dissect of trafficking and signalling mechanisms.

This work involves molecular techniques for construction of plasmid vectors expressing wild-type genes and mutant variants. We will examine post-translational modification of these proteins using well established methods, examining phosphorylation in the control of transport between cellular compartments, identify the sites within the protein that are modified and the important kinases involved by in vivo and in vitro techniques. We wil also examine insulin mediated control of transport and cleavage, linking this to insulin mediated phosphorylation.
In a second theme we will establish cell lines for regulated expression of wild-type and variant proteins and develop chromatographic purification schemes to identify interacting proteins by mass spectrometry. We will then develop antibodies and siRNA approaches to confirm interacting proteins and their role in transport of CREB3 proteins.

The Research Council guidance on impact and potential beneficiaries states that applicants are not expected to predict impact, but rather consider who might, potentially, benefit and how.

Who might benefit. The public in health and disease.
Why and how. This application is at a stage termed early discovery research in a pharmaceutical company setting. While one cannot predict the extent of the impact, there is a possibility of contribution to human health considering the pathways with which this application is concerned (e.g. triglyceride metabolism and bone formation). However by definition, while the most obvious ambition is for medical research, it has also the longest time frame and a complex pathway. We are asked to limit impact to the lifetime of the grant. Therefore at this stage the main contribution is in ensuring success of the research, its publication and its dissemination which determine further steps; being alert to opportunities and ensuring specialist advice in IP or commercial sponsorship as the work develops.

Who might benefit. The biotechnology sector.
Why and how. Many companies across human and animal sectors have interests apart from development of diagnostic or therapeutic entities. The pathways to Impact here are similar to but shorter than above. As an example, production of secreted biologicals. We have demonstrated that certain of the factors in this proposal increase secretory rates in cells, an activity which may be exploitable in applied settings. There are other examples. While one cannot predict impact in these fields, one needs to be aware of them and of the diverse interests in the sector. I am aware of such possibilities and the pathways through combinations of research, IP, licensing etc, for creating impact.

Who might benefit. Society and skills
Why and how. This research trains individuals in a broad range of skills, in addition to those required for specialised research, e.g., the transferable skills in computing and IT, statistics and data handling, problem solving, presentational and writing skills and more. While desirable to keep within research, such highly trained people make an Impact in other activities they may embark upon. The Pathway to this Impact requires a secure foundation in which to pursue the research, in-house or external training facilities if needed, mentorship and constructive evaluation. Imperial College makes available many such courses for its researchers, forums for discussion and for representation of its scientists and mechanisms to aid development for external opportunities. This Impact is achievable over the lifetime of the grant.

Who might benefit. Education sector
Why and how. We need to ensure supply of high quality students in the life sciences and medical research. We encourage engagement with schools and pupils and have dedicated events for investigators to promote their work. At an individual level, my laboratory accepts work experience students, stimulating and training the students with a real impact on their results and future careers. We routinely help supervise undergraduate students in projects or individual tutorials. We also give talks in schools, including primary schools or help teachers in promoting science.

Who might benefit. Science Communications, reporting and the media
Imperial College aims to create wide awareness of the benefits of research in science and medicine. I am listed as a researcher as a point of contact for science and news reporting. Imperial also produces professional materials to communicate research through a variety of routes and encourages investigators to promote their research directly to the public, for example using social media platforms, and engaging with audiences through events at Imperial and elsewhere. These routes have Impact in promoting research, creating informed commentators and engaging with various internal and external audiences.