Protein-protein interactions in recruitment of ubiquitylated proteins to the proteasome

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

The activities of many proteins are controlled by the addition of a small highly conserved protein called ubiquitin in a process called ubiquitylation. In particular, protein ubiquitylation plays an important role in controlling cell growth and division and in neurodegeneration. In cell cycle control, key proteins that regulate the cell cycle have to be degraded in a timely fashion to ensure appropriate progression through the replicative cycle. This is done by tagging the proteins with a chain of ubiquitins, linked in a specific fashion,to target them to the proteasome. The proteasome is a multi-protein complex that degrades proteins into short peptides and amino acids. Errors in this control pathway can lead to uncontrolled cell proliferation and ultimately to cancer. In many cases, proteins also have to be destroyed because they have lost their three-dimensional structure and cannot be allowed to accumulate in the cell. This protein damage happens throughout a cell?s lifetime as a result of exposure to certain cellular stresses and chemicals. In various neurodegenerative diseases including Parkinsons there is growing evidence that some of the proteins that are responsible for tagging misfolded proteins with polyubiquitin chains to target them to the proteasome do not function appropriately. This leads to an accumulation of certain misfolded proteins and is a feature of particular degenerate neuronal cell types. Although this research does not bear directly on designing potential therapeutics, it does aim to contribute to a greater understanding of how the polyubiquitin signal is recognised and how polyubiquitylated proteins are subsequently targeted to the proteasome. Our knowledge of these signalling pathways would be greatly assisted by knowing the structures of the ubiquitin-binding proteins and of their receptors at the proteasome in molecular detail. Protein structures can be determined using the techniques of X-ray crystallography and Nuclear Magnetic Resonance spectroscopy (NMR). The aim of the proposed research is to use both these techniques to characterise the regulation of and determine structures for the proteins that control the delivery of ubiquitylated proteins to the proteasome. Ultimately we may be able to exploit this knowledge for the treatment of disease.

Technical Summary

The Ubiquitin (Ub)-mediated protein degradation pathway (UPS) regulates many critical eukaryotic cellular processes that include the cell cycle, protein quality control and transcription. Dysfunction of the UPS has been associated with the progression of various human diseases, notably cancer and those of neurodegeneration. This project aims to combine biochemical, biophysical, structural and genetic approaches to further understanding of the mechanisms by which proteins modified by the addition of a Lys48-linked polyubiquitin (polyUb) chain are selectively targeted to the proteasome for degradation. Discrimination between mono-Ub and polyUb chains exhibiting different linkage types is essential to maintain fidelity in the different pathways regulated by ubiquitylation. Primarily through genetic experiments, a wide range of proteins has been identified that compose the pathways that target K48-linked polyubiquitylated proteins to the proteasome. S. pombe Pus1 is the ortholog of the human proteasomal subunit S5a, the first proteasomal Ub receptor to be identified. It has a C-terminal Ub-interacting motif (UIM), that interacts with Ub, and an N-terminal von Willebrand A (VWA) domain that interacts with the Mts4 and Mts3 subunits of the proteasome. Once the polyubiquitylated protein is bound to its proteasomal receptor it is unclear whether all proteasome substrates are processed through the same pathway or whether multiple parallel and /or alternative pathways exist. To begin to address this question, we have recently determined a low resolution structure of the Pus1 VWA domain and have shown, using Surface Plasmon Resonance methods, that the UIM of Pus1 interacts preferentially with Lys48-linked polyUb versus N-C linked linear polyUb chains. Employing X-ray, NMR and SPR methods we aim to elucidate the relationship between the structure and function of Pus1 and the proteasomal subunits to which it delivers its ubiquitylated cargo. Specifically we aim to (i) extend the resolution of our Pus1 structure to obtain atomic details; (ii) characterise the interactions of Pus1 with Mts4, SpRpn2 and Mts3; (iii) validate the functional significance of these interactions by in vivo studies and (iv) use the insights derived from these studies to identify complexes of Pus1 bound to Mts4 and Mts3 for structure determination. These are complex structural challenges but are fundamental to an understanding of how Ub is used as a protein post-translational modification for signalling within the cell.

Publications

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Trempe JF (2010) A new crystal form of Lys48-linked diubiquitin. in Acta crystallographica. Section F, Structural biology and crystallization communications

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Watson AF (2011) MDM2-p53 protein-protein interaction inhibitors: a-ring substituted isoindolinones. in Bioorganic & medicinal chemistry letters

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Anil B (2013) The structure of an MDM2-Nutlin-3a complex solved by the use of a validated MDM2 surface-entropy reduction mutant. in Acta crystallographica. Section D, Biological crystallography

 
Description Wellcome Trust Studentship
Amount £130,500 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2007 
End 09/2010
 
Description Characterisation of the ubiquitin-mediated protein degradation pathway 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution (i) Structure determination of various proteins in the ubiquitin-mediated protein degradation pathway (ii) biophysical and biochemical analysis and (iii) Generation of mutant constructs for genetic characterisation.
Collaborator Contribution (i) Provision of DNA sequences for protein expression. (ii) Genetic characterisation (iii) Expertise in the biology of the system aiding experimental design and direction
Impact (i) Structure determination of the proteasome 19S regulatory particle subunit Rpn12 (MS ID:22906049) (ii) Structure determination of Rpn10 and characterisation of its interactions with ubiquitin chains and the proteasome (MS ID: 20739285). (iii) Characterisation of the interaction of S. pombe Rhp23 with ubiquitin (MS ID: 23038266). Multidisciplinary collaboration involving yeast genetics, biophysical methods and X-ray crystallography.
Start Year 2006
 
Description Department Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Open Day posters describing the group's research. Tour of the facilities. Open Day well attended by sixth form students looking to study at Oxford.

An opportunity to inform sixth form science teachers that we welcome students for work experience placements.
Year(s) Of Engagement Activity 2013