Role of ISG15 in cancer
Interferon stimulated gene 15 (ISG15) is 165 amino acids in length and its protein is roughly 15kDa. ISG15 is a member of the ubiquitin-like family (Ubl) and shows homology to several regions of ubiquitin. The ISG15 protein was first identified in 1979 in mouse Ehrlich ascites tumour cells. ISG15 is upregulated by type 1 interferons, primarily α and β. ISG15 can also be induced by IFN regulated factors (IRFs). ISG15 has two different forms in a cell. The free form and the conjugated form. The free form has been shown to have anti-tumour effects whereas the conjugated form is generally involved in tumorigenesis. A higher concentration is found in primary tumours with the levels decreasing as the tumour progresses in stages. Therefore, ISG15 could be potentially used as an early detection diagnostic marker. ISG15 is normally only conjugated to a small number of proteins in a cell. It has been suggested that these proteins are localized to a specific functional cellular site or else the modification significantly alters the protein. ISGylation is a post-translational modification similar to ubiquitination. ISGylation is the process through which ISG15 can be conjugated to a target protein. The ISGylation process is thought to antagonize the ubiquitin pathway. Proteins which are selected for degradation via the ubiquitin/26S proteasome pathway can be stabilised by ISGylation and avoid degradation. If this occurs with proteins which normally exert an oncogenic effect, malignant growth can ensue. This is the case with proteins such as k-Ras. In this project, three different aspects of ISG15 were investigated; the role of type 1 interferons in ISG15 induction, targeted knock-down of ISG15 using CRISPR plasmids and single chain purification of ISG15. To investigate the IFN induction of ISG15, cells were transfected for varying lengths of time and then the expression levels of ISG15 were compared using Western blot analysis. The optimum time of INF transfection for ISG15 production is between 16 and 24 hours with no difference being seen for transfecting for a longer time period. The targeted knock-down of ISG15 using the CRISPR/CAS9 technique showed that the best area of the gene to target was the C-terminus as it was the only area to show a physiological change. CRISPR plasmids targeting the start of the coding exon (Exon 2) showed to have no effect. Following the single chain purification, the activity was tested using Elisas and showed to have a high affinity for pure ISG15.