INVESTIGATION OF THE FUNCTION OF NOVEL ISOFORMS OF THE EPITHELIAL CELL POLARITY PROTEIN DISCS LARGE 1

There are just over 300 PDZ (PSD95/DLG/ZO-1) domain-containing proteins (PDZ proteins) encoded by the human genome. PDZ proteins often function as “scaffolding proteins” and orchestrate the assembly of large signal transduction networks at specific sites within the cells, including at the plasma membrane. They have been shown to play important roles in diverse cellular processes including the establishment and maintenance of cell polarity, cell proliferation and cell trafficking. Deregulation of PDZ protein function has been linked to a number of human pathologies, including cancers, heart disease, hearing impairment and several different neuro-pathologies. Many of these pathologies impact on health in old age. A number of PDZ proteins have also been shown to be common targets of pathogenic viruses such as human papillomavirus (HPV)1.
The multi-PDZ domain-containing protein discs large 1 (DLG1) is an essential component of one of epithelial polarity complexes and is the necessary for maintenance of apicobasal cell polarity. DLG1 is also involved in the organization of neuronal synapses and in regulation of asymmetric cell division1.
Alternative RNA splicing of PDZ proteins is common and represents a mechanism of increasing the functional diversity of this important family of proteins. To date, a number of alternatively spliced variants of DLG1 have been characterized that use alternative exons and these spliced isoforms contain small insertions within the coding sequence of the polarity protein. The alternative splicing of DLG1 has been shown to impact on DLG1 function, through differential targeting to specific cellular sites and binding of protein interacting partners2; however, we are still lacking a considerable amount of information about DLG1 function and how it is regulated.
We have identified in physiologically relevant skin models, novel alternatively spliced variants of DLG1 that contain a previously un-described exon. Predictive analysis of the proteins encoded by the novel DLG1 isoforms indicated that some produce truncated proteins that lack the C-terminal domain of DLG1. Therefore, we have a number of important questions:(i) What are the specific functions of novel alternatively-spliced DLG1 isoforms and are they cell context-dependent? (ii) With respect to their functions in keratinocytes, are they involved in the process of growth and differentiation of epithelial differentiation? (iii) In the case of those isoforms that are predicted to encode truncated forms of DLG1, do they regulate DLG1 function by using a dominant-negative mechanism? (iv) What is(are) the mechanism(s) that regulates alternative RNA splicing of DLG1 in epithelial cells?
To address these questions, the student will clone the different isoforms into suitable expression systems and characterize their expression (e.g. protein levels, cellular distribution, influence of epithelia differentiation) in primary keratinocytes. By using this system they will also investigate the effect of the novel isoforms on DLG1 function, including its role in epithelial differentiation by using organotypic raft cultures. Function will also be explored further through the isolation and identification of interaction partners by using of liquid chromatography-tandem mass spectroscopy of co-immunoprecipitated proteins. Since we have shown that the novel isoforms are up-regulated in cells undergoing HPV DNA replication, we have available an excellent keratinocyte-based model system to investigate the mechanism(s) that regulate alternative splicing of DLG1.

Key experimental skills involved:
The student will undertake a wide range of molecular and cell biology techniques, biochemical methodology, as well as “omics”. Specifically, the student will acquire the key skills in primary epithelial cell culture and organotypic raft culture of keratinocytes to generate three-dimensional models of differentiated epithelia, fluorescence and live cell imaging, DNA cloning and mutagenesis. The student will also become proficient in proteomics and the bioinformatics analysis of the datasets collected.