 |
| Dr Gabriela Alexandru |
| E: g.alexandru@dundee.ac.uk |
| T: 44 1382 388591 |
| F: 44 1382 388500 |
Dr Gabriela Alexandru
Research
Exploration of the p97 and UBX-Protein Interaction Networks and Identification of Novel p97 Targets
Overview: The general objective of the research in my laboratory is to understand the function of p97 and its co-factors in human cells, with emphasis on their role in regulating protein degradation.
Ubiquitin-dependent protein degradation is not simply involved in the recycling of amino acids from misfolded proteins, but, more importantly, it represents an essential mechanism for modulating the levels of key regulatory proteins. In the past few years, various ubiquitin-receptors (UbR in Fig.1) have been identified that participate in processing and/or delivery to the proteasome of ubiquitylated substrates [1]. Among them, p97 is an ATP-dependent molecular chaperone that plays a role in diverse cellular activities, ranging from quality control pathways, such as the endoplasmic reticulum-associated protein degradation (ERAD), to homotypic membrane fusion and cell cycle regulation [2]. The role of p97 in regulating ubiquitin-dependent protein degradation has been explored mainly in the context of ERAD, where p97 provides the driving force for protein retro-translocation from the ER into the cytosol [3].
Many p97 functions involve recognition of ubiquitylated protein substrates and this is largely mediated by p97 co-factors. We analyzed p97 immunoprecipitates from human cells by shotgun mass spectrometry and identified eight p97 binding partners (shown in red in Fig. 2), all having an UBX domain at their C-terminus. We subsequently confirmed that all thirteen mammalian UBX proteins form independent complexes with p97. The biological role for most of these proteins is currently unknown, but it is likely that each of them specifies a different p97 function. To test this hypothesis, we performed comparative mass spectrometry analysis of UBX-protein immunoprecipitates searching for interactions specific to a single UBX protein.
We then focused on one UBX-protein, UBXD7, and explored the biological significance of its interaction with the master regulator of cellular response to hypoxia, HIF1-alpha. p97 is required for efficient degradation of HIF1-alpha during normoxia and UBXD7 is the p97 co-factor that recruits HIF1-alpha to the p97 complex [4]. Our proteomic studies predict that HIF1-alpha is just the tip of the iceberg and many other ‘soluble’, non-ERAD substrates of p97 will follow.
Part of the research in my laboratory relies on the proteomics datasets we obtained previously. Further proteomic analyses will be performed using SILAC (stable isotope labelling by amino acids in cell culture) and the state of the art mass spectrometry facility at SCILLS. These will aim to identify specific substrates for individual UBX-domain cofactors of p97, as outlined below.
A. Exploration of the UBX-Protein Interaction Networks and Identification of Novel p97 Targets
1. Novel ubiquitin-dependent substrates of p97
Five of the UBX-domain proteins, the UBA-UBX proteins, contain an UBA (ubiquitin-associated) domain at the N-terminus (Fig. 2), which enables them to bind ubiquitylated proteins. Moreover, through mass spectrometry analysis, we identified numerous E3 ubiquitin-ligases that interact with UBA-UBX proteins [4] (Fig. 3). Thus, UBA-UBX proteins are likely to target a large spectrum of substrates carrying an ubiquitin modification.
HIF1-alpha is so far the only non-ERAD substrate of p97 that has been linked to a particular UBX protein. The identification and characterization of other UBA-UBX protein – substrate pairs will not only expand our knowledge of p97 function and specificity, but will also allow a better understanding of the p97 mechanism of action towards substrates that do not require retro-translocation from the ER.
2. Targets for UBX-only p97 Co-Factors
Due to the lack of an UBA-domain, the eight UBX-only proteins do not have the ability to bind ubiquitin, which might restrict their substrate specificity. Furthermore, because several UBX-only proteins are preferentially expressed in particular tissues, it is possible that their function is not only restricted to a particular target, but also to a certain cell type. Mass spectrometry analysis of UBX-only protein immunoprecipitates will be performed using cells in which they are normally expressed and more likely to interact with physiological partners.
The study of UBX-only p97 co-factors is of particular interest, because it has the potential to expand our knowledge of ubiquitin-independent p97 functions.
B. The Molecular Defect of Disease-Causing p97 mutants
Mutations in p97 are the underlying cause for the syndrome of inclusion body myopathy with Paget’s disease of the bone and frontotemporal dementia (IBMPFD), a progressive and ultimately lethal autosomal dominant disorder characterized by accumulation of protein aggregates [5]. Most of the IBMPFD mutations map to the N-terminus of p97, known to be required for the interaction with co-factors like NPL4, UFD1 and the UBX-domain of p47 [6]. It is therefore enticing to investigate whether the impaired activity of disease-causing p97 might be due to a defect in binding one or more of its co-factors. Comparative mass spectrometry analysis of wild type and mutant p97 immunoprecipitates will be performed, searching for differences in their spectrum of protein interactions. Besides providing important insights into the cause of IBMPFD, these experiments might further the understanding of other neurodegenerative disorders characterised by the accumulation of ubiquitin-positive aggregates.
References
- Elsasser, S. and Finley, D. (2005). Delivery of ubiquitinated substrates to protein-unfolding machines. Nat Cell Biol. 7, pp. 742-749.
- Ye, Y. (2006). Diverse functions with a common regulator: Ubiquitin takes command of an AAA ATPase. J Struct Biol. 156, pp. 29-40.
- Ye, Y., Meyer, H.H. and Rapoport, T.A. (2001). The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, pp. 652-656.
- Alexandru, G., Graumann, J., Smith, G.T., Kolawa, N.J., Fang, R. and Deshaies, R.J. (2008). UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover. Cell 134, pp. 804-816.
- Watts, G.D., Wymer, J., Kovach, M.J., Mehta, S.G., Mumm, S., Darvish, D., Pestronk, A., Whyte, M.P. and Kimonis, V.E. (2004). Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet. 36, pp. 377-381.
- Yeung, H.O., Kloppsteck, P., Niwa, H., Isaacson, R.L., Matthews, S., Zhang, X. and Freemont, P.S. (2008). Insights into adaptor binding to the AAA protein p97. Biochem Soc Trans. 36, pp. 62-67.
University of Dundee