Structure and dynamics of oligomeric intermediates in amyloid assembly

Amyloidosis is associated with devastating diseases including Alzheimer's, Parkinson's and type II diabetes. Although the proteins responsible for these diseases vary widely in their amino acid sequences and structure of the monomeric precursor, they all form insoluble polymeric structures termed amyloid fibrils, which share a common, distinctive morphology with a so-called cross-beta structure. The assembly pathway for many proteins is thought to start with the protein monomer unfolding partially or completely from its specific, native 3D structure. Once unfolded, the monomer is able to polymerise, or self-assemble, into the distinctive, long, twisted fibrils that accumulate in various organs of the body and are associated with amyloid disease. However, the precise pathways of assembly, and the nature of oligomeric intermediates, (one or more of which are thought to be the culprits of the toxicity associated with amyloid disease), remain unknown. Here we propose to characterise these oligomers in unprecedented detail, thus mapping the pathway of amyloidosis in molecular detail and identifying possible oligomeric targets for future therapeutic remedies. To address these issues, we will work on the amyloidogenic protein, beta2-microglobulin (beta2m), a protein which forms amyloid fibrils in all patients undergoing long term renal dialysis. The applicants have gained a wealth of experience with this protein over several years and hence it is an ideal system on which to carry out the proposed research. In addition to new insights into beta2m fibril assembly, the results generated and protocols developed will have direct importance and relevance for all amyloid systems. To paint a comprehensive picture of the amyloid assembly pathway we propose to employ a new and exciting combination of two techniques: ion mobility spectrometry and mass spectrometry. In a single, rapid experiment we are now able to detect, quantify and individually characterise transient intermediates within heterogeneous ensembles in real-time during fibril assembly. Building on a mass of preliminary data that demonstrate the powers of this approach we will measure (i) the molecular mass, (ii) the cross-sectional area (which is determined by shape), (iii) the stability (using collision-induced dissociation and subunit exchange experiments) and (iv) the ligand binding capability of different, individual oligomeric species. We will also use in-house developed molecular modelling programs to compare the experimental data with theoretically plausible structures. By combining these experiments with protein engineering, in which the side-chains of individual amino acids will be altered one by one, and other biochemical and biophysical analyses, we will determine which residues are responsible for specific oligomers being formed, and will thereby map the importance of each oligomer in fibril assembly. We will also use the methods developed to compare assembly under physiological and non-physiological conditions, so as to discern the heterogeneity of the assembly landscape. Finally, we will study the binding of a small molecule ligand that we have recently identified as novel, potent inhibitor of beta2m amyloid assembly. The consequence of ligand binding on the population, structure, and stability of individual oligomeric species will be assessed, providing new insights into how a small ligand can comprehensively arrest the self-assembly of a large protein subunit. Together these experiments will provide unprecedented detail into the fundamental mechanisms of amyloid assembly and will greatly enhance our understanding of this unwanted biological phenomenon. Moreover, by characterising the structural and ligand binding properties of individual oligomers, we aim to identify, for the first time, individual molecular species as the targets for future therapeutic intervention.

Amyloid fibril formation is associated with many devastating diseases. The proteins responsible for different amyloid disorders are unrelated in sequence/structure, their common feature being the ability to form amyloid fibrils with a common cross-beta architecture. Despite the importance of amyloid disorders in today's ageing population, the molecular mechanisms of amyloidosis remain elusive. In particular, the oligomeric intermediates in assembly, some of which have been linked with the toxicity associated with amyloid disease, have not been characterised at the molecular level, leaving open the key questions of the role of different oligomers in amyloid assembly and hence the identification of new, defined targets for intervention in amyloid disease. Here we propose to exploit electrospray ionisation-mass spectrometry coupled to travelling wave ion mobility spectrometry (ESI-TWIMS-MS) to provide new insights into the conformational properties of specific oligomeric intermediates in amyloid assembly. This approach has the unique capability of separating co-populated isobaric and oligomeric biomolecules of the same m/z in real-time while retaining their tertiary and quaternary structure. By focusing on the naturally amyloidogenic protein, beta2-microglobulin (beta2m), we will use ESI-TWIMS-MS, complemented by protein engineering experiments and other biophysical techniques, to determine the mass, cross-sectional area, stability and longevity of individual oligomeric intermediates in amyloid assembly commencing from either an acid unfolded state (pH 2.5) or a native-like conformer (pH 7.0) of the same protein. In addition, following on from an exciting recent discovery, we will study the binding of a small molecule we have identified as a potent inhibitor of beta2m amyloid assembly when added in the lag time of assembly, with the aim of identifying a specific oligomer target for future therapeutic design.

Scientific impact: Protein folding, mis-folding and self-aggregation have both biochemical and medical importance. The proposed project will have wide scientific impact as well as generating a wealth of new and important information concerning the oligomeric intermediates associated with amyloid disease, with which it may be possible to counter such protein-related diseases in the future. This is a challenge of biological importance; the results will be of interest to a wide spectrum of scientists, pharmacists and medical researchers in both academia and industry. Industrial impact: Our long-term collaboration with Waters UK Ltd (mass spectrometry manufacturers), which has resulted in three BBSRC/CASE PhD studentships to date, confirms their continued interest in developing mass spectrometry to solve biological problems. Similarly, our collaboration with the Laboratory of the Government Chemist (National Measurement Institute), which currently funds a PhD student with AEA, and our invited involvement in their Chem-Bio Metrology Project 'Ion Mobility Spectrometry for Security and Pharmaceutical Applications' confirms the importance of ion mobility calibrations and measurements. We also have close contact with several pharmaceutical companies (Pfizer, AstraZeneca) who have donated equipment to us, and who have medicines aimed at ageing-associated amyloid diseases. Supporting knowledge and technology translation: Both AEA and SER speak regularly at national and international scientific conferences. Additionally, AEA was a plenary speaker at the 1st Global AstraZeneca Mass Spectrometry Meeting in 2009 and presents regularly at manufacturers' meetings (Waters, Advion Biosciences). AEA and SER won the 2009 ASMS Ron Hites prize for outstanding research which resulted in press releases by Waters and by ASMS. In addition, SER's recent research into amyloid fibril toxicity (funded by the Wellcome Trust and a BBSRC DTG studentship) has been highlighted on the front page of the BBSRC website (6.12.2009), and chosen as paper of the week in J. Biol. Chem., illustrating the importance of the research in this area and the pro-active nature of the researchers and the University in promoting its research findings. Each Faculty of the University of Leeds has a dedicated Knowledge Transfer (KT) Director with a budget dedicated to facilitating KT activities. Intellectual property that has arisen from Astbury Centre for Structural Molecular Biology (ACSMB) research projects has been protected in conjunction with the University's Enterprise and Innovative Office and spin-off companies have arisen. AEA is the ACSMB Industry Liaison Office and held a recent Open Day for Industry to which representatives from pharmaceutical and biotechnological industries attended with a view to creating stronger links with the ACSMB. Delivering highly skilled people: The proposed project will train a PDRF in cutting edge technology and ensure their future successful career development, whether in academia or industry. Over the past few years, highly skilled post-graduate and post-doctoral fellows from the laboratories of both AEA and SER have gone on to post-doctoral fellowships and lectureships in other universities, and positions in industry. Public engagement: ACSMB members have a strong track record of engagement with the general public, including workshops and science fairs to inspire school children to study science, student placements in research laboratories, talks at conferences for school teachers and in schools, and authorship of articles for sixth-formers. ACSMB works closely with CampusPR to promote its science effectively to audiences outside academia, in print (newspapers, popular scientific journals), on-line (e.g. BBC, Daily Telegraph, industry-focused websites) and on the radio. Thus ACSMB vigorously increases the impact of its members' research through engaging with industry, government bodies and the general public.