Cullin 7 (Cul7) is a member of the cullin protein family that is emerging as a complex anti-apoptotic player in tumourigenesis. We hypothesize that by determining the mechanism through which Cul7 can protect against specific forms of cell death, we will uncover novel molecular pathways important in cancer. We aimed to address mechanism by evaluating which domains within Cul7 are important for its activity. Thus, we have introduced mutations in each of the Cul7 domains and asked whether any of these has an effect on the ability of Cul7 to inhibit cell death. To be able to detect even subtle affects of mutation, we required that mutants be assessed in appropriate experimental systems where ectopic wild-type Cul7 could robustly inhibit death compared to vector controls. We screened multiple cell lines and agonists, and have now indentified conditions in U2OS and SHEP cell lines in which our mutants can be evaluated.

Cullin 7 (Cul7) is a member of the cullin protein family that is emerging as a complex anti-apoptotic player in tumourigenesis. We hypothesize that by determining the mechanism through which Cul7 can protect against specific forms of cell death, we will uncover novel molecular pathways important in cancer. We aimed to address mechanism by evaluating which domains within Cul7 are important for its activity. Thus, we have introduced mutations in each of the Cul7 domains and asked whether any of these has an effect on the ability of Cul7 to inhibit cell death. To be able to detect even subtle affects of mutation, we required that mutants be assessed in appropriate experimental systems where ectopic wild-type Cul7 could robustly inhibit death compared to vector controls. We screened multiple cell lines and agonists, and have now indentified conditions in U2OS and SHEP cell lines in which our mutants can be evaluated.

3-M syndrome (named after three authors who first described the condition) is an autosomal recessive condition characterised by pre- and post-natal growth impairment, facial dysmorphism and radiological features (slender long bones and tall vertebral bodies). It is caused by loss of function mutations in the Cullin 7 (CUL7) and Obscurin-like 1 (OBSL1) genes. CUL7 is a protein involved in ubiquitination (the process of targeted protein degradation) and OBSL1 is a putative cytoskeletal adaptor protein. The mechanisms through which loss of function mutations in OBSL1 or CUL7 lead to growth impairment is unclear but previous work suggests impaired placental function and altered insulin-like growth factor 1 (IGF-1) signaling as possibilities. The overall aim of this study was to elucidate the mechanisms underlying growth impairment in 3-M syndrome. Initially phenotypic data was collected on a cohort of patients and a genotype-phenotype comparison was undertaken. Skin fibroblast cell lines were derived from four patients with 3-M syndrome and used to study growth hormone (GH) and IGF-1 signal transduction, cell proliferation and apoptosis. Subsequently a hypothesis generating approach to identify novel mechanisms underlying 3-M growth impairment was undertaken in whole transcriptome and metabolomic studies. In addition an animal model using morpholino oligonucleotide mediated knock down of OBSL1 in Xenopus tropicalis was developed to study the effects on growth in a non placenting vertebrate to determine if the growth impairment seen in 3-M syndrome is independent of placental function. Cell proliferation was reduced in 3-M fibroblasts while apoptosis was not different from controls. No differences in GH signal transduction were identified but reduced activation of AKT following IGF-1 stimulation was identified in 3-M fibroblast cell lines. IGF2 was identified as the top downregulated probeset in 3-M fibroblasts compared to control in the whole genome transcriptome analysis. Metabolomic changes related to energy metabolism were identified in 3-M syndrome fibroblasts. Knock down of xtOBSL1 using two independent morpholinos resulted in growth impairment at embryonic stage 50, suggesting the growth impairment seen is at least in part independent of placental function. These studies suggest impaired placental function is not a key component of the growth impairment in 3-M syndrome. Impairment of IGF-1 signal transduction and IGF2 silencing are likely to contribute to the growth impairment in 3-M syndrome. The mechanisms relating to this IGF2 silencing require further studies.

A convincing body of evidence suggests that ubiquitination and the ubiquitin proteasome degradation pathway play a key role in neoplastic transformation. Ubiquitination, as post-translation modification, is involved both in functional regulation and degradation of specific cellular targets known as proto-oncogenes and tumor suppressors. Oncogenic viral proteins interact both with proto-oncoproteins and tumor suppressors leading to the modulation of their cellular function by several mechanisms including ubiquitination. Interestingly, viral oncoproteins themselves can also be regulated by this post-translation modification. Additionally, viruses can assemble their own E3 ligases or regulate the activity of cellular E3 ligases. E3 ligases, involved in the final step of the ubiquitination process, are the enzymes that provide the specificity for the interaction with target substrates by the means of a large number of proteins. Recent studies on the potential correlation between viral infection and oncogenesis, have addressed the emerging role of the ubiquitination system as a possible mediator for cancer transformation. In this scenario we hypothesized that JCV T-antigen may interfere with the ubiquitination system and we investigated a possible interaction between JCV T-antigen and the E3 ligase Cul7. To prove our hypothesis we performed co-immunoprecipitation and co-immunofluorescence experiments using the glioblastoma cell lines HJC12, U87MG and HJC5. Our results indicate that JCV T-antigen and Cul7 interact in the cytoplasmic compartment. In addition, JCV T-antigen stabilizes Cul7. These observations suggest that JCV T-antigen can modulate Cul7 E3 ligase activity leading to oncogenesis. Further study addressing the biological significance of this interaction will decipher the cellular processes modulated by JCV T-antigen and Cul7 and will indicate new avenues for therapeutic intervention. / Biology