Establishment of human OCT4 as a putative HSP90 client protein: a case for HSP90 chaperoning pluripotency
- Authors: Sterrenberg, Jason Neville
- Date: 2015
- Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
- Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
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The role of Stress Inducible Protein 1 (STI1) in the regulation of actin dynamics
- Authors: Beckley, Samantha Joy
- Date: 2015
- Subjects: Heat shock proteins , Molecular chaperones , Actin , Microfilament proteins , Cell migration , Adenosine triphosphatase , Metastasis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193941 , vital:45409
- Description: Stress-inducible protein 1 (STI1) otherwise known as Hop (Hsp70/Hsp90 organising protein) is a highly conserved abundant co-chaperone of the Hsp70 and Hsp90 chaperones. STI1 acts as an adapter protein, where it regulates the transfer of protein substrates from Hsp70 to Hsp90 during the assembly of a number of chaperone-client protein complexes. The role of STI1 associating independently with non-chaperone proteins has become increasingly prominent. Recent data from colocalisation and co-sedimentation analyses in our laboratory suggested a direct interaction between STI1 and the cytoskeletal protein, actin. However, there was a lack of information on the motifs which mediated this interaction, as well as the exact role of STI1 in the regulation of cytoskeletal dynamics. Two putative actin binding motifs, DAYKKK (within the TPR2A domain) and a polyproline region (after the DP1 domain), were identified in mammalian STI1. Our data from in vitro interaction studies including surface plasmon resonance and high speed co-sedimentation assays suggested that both TPR1 and TPR2AB were required for the STI1-actin interaction, and peptides corresponding to either the DAYKKK or the polyproline motif, alone or in combination, could not block the STI1-actin interaction. Full length mSTI1 was shown to have ATPase activity and when combined with actin an increase in ATPase activity was seen. Ex vivo studies using STI1 knockdown shRNA HEK293T cells and non-targeting control shRNA HEK293T cells showed a change of F-actin morphology as well as reduction in levels of actin-binding proteins profilin, cofilin and tubulin in the STI1 knockdown cells. These data extend our understanding of the role of STI1 in regulating actin dynamics and may have implications for cell migration. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2015
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