An investigation into the interacting partners of the ubiquitin ligase, SIAH1, and the tumour suppressor protein, ASPP1

Abstract

Selective protein degradation is a crucial control mechanism which impacts nearly every aspect of eukaryotic cell biology. It is achieved via the ubiquitin-proteasome pathway whereby multimers of ubiquitin are conjugated to substrate proteins and subsequently recognised and degraded by the proteasome. The specificity of substrate recognition is provided by E3 ubiquitin ligase enzymes. Seven in Absentia homologue-1 (SIAH1) is a highly conserved E3 ubiquitin ligase which recognises and binds to an array of substrate proteins facilitating their ubiquitination and degradation. The identification of various SIAH protein substrates has revealed that SIAH1 and its family members play a role in a number of cellular processes including cell-cycle regulation, cell differentiation, and apoptosis. Previous studies also revealed that SIAH1 has a novel role in the testes as removal of the equivalent gene in mice (Siah1a) results in male sterility. Despite the numerous studies which have led to the identification of various SIAH substrates, how these proteins and their stability relates to the observed Siah1a mutant phenotype of male sterility remains unclear. Therefore, to better understand SIAH1 function in the testes, a number of SIAH1 interacting proteins identified in a yeast 2-hybrid screen of a human testes cDNA library were investigated. Primarily, experiments focused on finding out whether or not any of these proteins were targets for SIAH1-mediated degradation. Two prime candidates were further investigated including NELF-A, a transcriptional regulator of developmental control genes, and ASPP1, a p53 interacting protein which promotes apoptosis. Further investigation into the SIAH1:NELF-A interaction confirmed that a genuine molecular interaction occurred in vitro. Transient transfection assays and analysis of NELF-A protein in Siah1a-/-Siah2-/- fibroblasts revealed that SIAH proteins predominantly target ectopically expressed NELF-A protein for degradation and analysis of NELF-A regulation by the murine family of Siah proteins revealed that the closely related Siah1a protein, Siah1b, has diverged in function. Consistent with SIAH1’s role in apoptotic regulation, SIAH1 was found to regulate the stability of ASPP1 and its homolog ASPP2. To better understand the downstream consequences of SIAH1- mediated degradation of ASPP1 and to shed light on ASPP1 function, protein interaction screens were carried out to identify major ASPP1-binding protein partners. A diverse set of interacting partners were identified implying ASPP1 is a multifunctional protein involved in a number of cellular pathways.