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Structural and functional analysis of the proteasome inhibitor PL31 and its interaction with the F-box protein Fbxo7.

Kirk, R.J.; (2005) Structural and functional analysis of the proteasome inhibitor PL31 and its interaction with the F-box protein Fbxo7. Doctoral thesis , University of London. Green open access

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Abstract

This thesis describes the structural and functional analysis of two related proteins, PI31 and Fbxo7, both of which act within the ubiquitin-proteasome system. This system is involved in a wide variety of cellular processes. Proteins modified with ubiquitin are often targeted for degradation by the proteasome, an ATP-dependent multi-subunit complex. Specificity for ubiquitin transfer is controlled by the E3 ubiquitin ligases. The multi-subunit SCF E3 ubiquitin ligases use their F-box domain protein to engage substrates. Analysis of possible binding partners of the novel F-box protein Fbxo7 using an affinity pull-down in Jurkat cell lysates identified PI31, a putative proteasome inhibitor. Fbxo7 was also shown to bind Skpl, Cullinl and Rbxl inferring that it forms part of a functional E3 ubiquitin ligase. Crystallisation of the Fbxo7-Skpl complex yielded poorly diffracting crystals with a maximum resolution of 8A. The PI31 N-terminal domain (NTD) shows significant sequence similarity to a globular domain of Fbxo7. The PI31-NTD structure was obtained at 2.64A by MAD phasing after introducing an additional methionine to engineer a non-centrosymmetric selenium substructure. The structure reveals a homodimer of novel a/p topology, which we define as the FP (Fbxo7 - PI31) domain since its distribution is limited to Fbxo7 and PI31 proteins in higher eukaryotes. Biophysical analysis of structure-based mutations identified the relevant residues in the homodimer interface by confirming the production of a monomeric form of PI31. Further mutations also revealed the binding surface between Fbxo7 and PI31 by using isothermal calorimetry. Fbxo7 and PI31 were shown to interact in vivo by co-immunoprecipitation and yeast two-hybrid screens using either PI31 or Fbxo7 as bait. Fbxo7 was shown to ubiquitinate FLAG-tagged PI31 in vivo. Abolishing the interaction between Fbxo7 and PI31 by mutations in PI31 prevents this ubiquitination. A model for the interaction between PI31 and Fbxo7 is presented.

Type: Thesis (Doctoral)
Title: Structural and functional analysis of the proteasome inhibitor PL31 and its interaction with the F-box protein Fbxo7.
Identifier: PQ ETD:593054
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > CRUK Cancer Trials Centre
URI: http://discovery.ucl.ac.uk/id/eprint/1445730
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