Molecular, cellular and functional characterisation of novel proteasomal proteins hDDI1 and hDDI2

Muslim, Mohammed Dzaidenny (2012). Molecular, cellular and functional characterisation of novel proteasomal proteins hDDI1 and hDDI2 PhD Thesis, School of Medicine, The University of Queensland. doi:10.14264/uql.2015.907

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Author Muslim, Mohammed Dzaidenny
Thesis Title Molecular, cellular and functional characterisation of novel proteasomal proteins hDDI1 and hDDI2
School, Centre or Institute School of Medicine
Institution The University of Queensland
DOI 10.14264/uql.2015.907
Publication date 2012-10-10
Thesis type PhD Thesis
Supervisor Nathan Subramaniam
Daniel Wallace
Total pages 212
Total colour pages 16
Total black and white pages 196
Language eng
Subjects 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
060199 Biochemistry and Cell Biology not elsewhere classified
060103 Cell Development, Proliferation and Death
Abstract/Summary The ubiquitin-proteasome system plays an important role in a wide variety of cellular processes including gene regulation, cell cycle and DNA damage sensing/repair. The degradation of cellular proteins through the ubiquitin-proteasome pathway is a highly complex and tightly regulated process. Recently, much research has focused on a class of proteins known as the UBA-UBL domain proteins which contain two functional domains related to ubiquitin: the ubiquitin-associated (UBA) and the ubiquitin-like (UBL) domains. The ability of these proteins to interact with both ubiquitin and the proteasome through both domains generated the interesting proposition whereby proteins belonging to this group may act as ‘shuttle factors’ bridging ubiquitinated substrates to the proteasome for degradation. These “shuttle factors” may thus contribute to the level of specificity and regulation to the complex mechanism of selective protein degradation through the ubiquitin-proteasome system. The yeast DNA Damage Inducible gene 1 product or Ddi1 belongs to the UBA-UBL class of proteins. We have identified the human homologues of this protein which we have designated, hDDI1 and hDDI2. Our characterisation studies have indicated a putative function for the latter protein in the regulation of the tumour suppressor protein, p53, through the ubiquitin-proteasome pathway. Specific antibodies against both hDDI and hDDI2 were raised and utilised in immunoprecipitation and western blotting experiments. Utilising these antibodies, we have shown that the expression of hDDI1 is expressed at very high levels in the testis. hDDI2 is ubiquitously expressed. Analysis of human cell lines at the protein and mRNA level also showed that hDDI2 is expressed in all of the cell lines examined. As hDDI1 is not present to any detectable level in human cell lines, our subsequent studies have focussed on the more widely expressed hDDI2. Given that yeast Ddi1 interacts with the proteasome, we examined the interaction between hDDI2 and the proteasome. Our results showed that hRPN13, a subunit of the 26S proteasome, can be co-immunoprecipitated in the same complex as endogenous hDDI2 in U2OS cells and that this was a direct interaction was shown through in vitro binding studies. We employed siRNA-mediated knockdown of hDDI2 and studied its phenotype in cells. Using a real time cell analyser platform to measure cell growth, we showed that vi knockdown of endogenous hDDI2 led to growth retardation in U2OS cells when compared to control cells. Additionally, cells depleted of hDDI2 displayed reduced cell survival after exposure to DNA damage. We studied the effect of hDDI2 knockdown on the proteasome-mediated degradation of the tumour suppressor protein, p53, a well studied substrate of the ubiquitin-proteasome pathway. Previous studies have shown that the ubiquitination of p53 leads to its subsequent degradation by the proteasome. We found that knockdown of endogenous hDDI2 caused an acceleration in the degradation of p53 which led to the dysregulation of p53 levels in U2OS cells. The p53 protein plays an important role in the DNA damage pathway. Upon DNA damage, p53 levels are stabilised and activated to induce downstream DNA damage repair genes. In our study, we found that the depletion of endogenous hDDI2 before exposure of U2OS cells to DNA damage abrogated the stabilisation of p53. Lastly, we performed a proteomic analysis of hDDI2 interacting proteins in mouse liver tissue and U2OS cell lysates using anti-DDI2 antibodies. Several putative interacting proteins including different subunits of the proteasome were identified. Our findings suggest a role for hDDI2 in the regulation of the tumour suppressor protein, p53 through the ubiquitin-proteasome pathway. We propose a model by which the interaction of hDDI2 with the proteasome serves as an important regulator in maintaining the cellular levels of p53. Future work aims to investigate the mechanisms that are involved in the regulation of p53 by hDDI2 and also identify other factors that are involved in this regulation.
Keyword Ubiquitin-proteasome pathway
DNA damage

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Created: Wed, 10 Oct 2012, 11:21:26 EST by Mr Mohd Haji Muslim on behalf of Scholarly Communication and Digitisation Service