Deciphering the function of intrinsically disordered protein regions in a cellular context

Proteins carry out the chemical reactions necessary for life, and are used as building blocks to assemble key components of cells, giving them shape and structural integrity. During a cell's life cycle, different proteins are produced as needed and then recycled when they have finished their work. To perform their jobs, proteins may themselves undergo chemical modifications, interact with other proteins and adopt a variety of different shapes. Our understanding of protein shape, structure and function has been enormously useful in furthering our molecular understanding of life, leading to successful drug-discovery efforts, methods to improve crop production and other applications with economic and societal benefits. While most proteins adopt a regular 3D shape, it is now accepted that large sections of many proteins termed intrinsically disordered regions (IDRs) have no fixed shape. These "shape-shifting" properties allow the proteins that contain them to perform different jobs at different times and in different parts of the cell by dynamically adopting different shapes in response to their environment. To truly understand the "molecular rules of life", it is therefore necessary to understand how the structures of these "shape-shifters" changes with time, how this influences what other proteins they interact with, how this impacts on the healthy/unhealthy cells life-cycle and ultimately how to control these properties using chemistry.

In this research we will study a protein that plays an essential role in the cells life-cycle (Aurora-A) e.g. in cell-division, a process that becomes defective in cancer making it a focus of anticancer drug discovery efforts that have not yet been successful. Aurora-A fulfils different jobs at different times and in different parts of the cell by interacting with multiple different "shape-shifting" proteins.

We will use an integrated and state-of-the-art chemical and biological approach to characterise when, where and which interactions between shape-shifting proteins and Aurora-A define its biological function. In doing so, we will identify methods to switch off the interactions between Aurora-A and specific shape-shifters, which can be used to further understand the functional role of these proteins and provide starting points for drug discovery. About a third of human proteins are thought to have an intrinsically disordered region, and our study will help biologists to investigate the properties and roles of these poorly-understood proteins. In the longer term, the ability to manipulate "shape-shifting" proteins will open up a new route to developing medicines to treat a wide range of diseases.

This proposal aims to understand and manipulate the dynamic features of order-disorder transitions in intrinsically disordered regions (IDRs) of proteins at the molecular scale, and to identify tools that modulate the interactions between Aurora-A and IDRs in cells. This requires a unique multidisciplinary approach and large collaborative effort to address our objectives. The sLola proposal is organised through three interconnected work-packages that deliver the necessary technical capabilities as follows:

WP1 - Structural Biology and Biophysics: We will carry out in vitro analyses of Aurora-A/IDR interactions, together with analyses of order-disorder transitions (using appropriate methods incl. NMR, single-molecule fluorescence spectroscopy, X-ray crystallography), to guide inhibitor design and understand dynamic structural changes for Aurora-A/IDR interactions in the context of multicomponent scaffolding complexes.

WP2 - Making Reagents: We will map Aurora-A interaction sites on IDRs using peptide arrays, tag-transfer photo-crosslinking and chemical proteomics. We will also use a suite of established capabilities including constrained peptides and non-antibody binding proteins (incl. Affimers) to identify novel ligands that target specific Aurora-A/IDR interactions in cells.

WP3 - Functional Analyses in Cells: We will use gene editing and additional methods in human cell lines to knockout and/or modulate the dynamics of Aurora-A/IDR interactions. We will harness these modified and other cell lines together with assets developed in WP1-2 to probe the functional role of Aurora-A/IDR interactions and their modulation using transcriptomics and an array of established phenotypic and functional assays in cells using high-resolution microscopy.

This will allow correlation of Aurora-A functions/interactions with the dynamics of cellular processes (e.g. duration of different phases of mitosis and spindle formation, microtubule nucleation, ciliogenesis).

Interactions involving IDRs are common in the cellular systems and signalling pathways associated with cancer, diabetes, cardio-vascular and neurodegenerative diseases, and in plants, where for instance IDRs are involved in adaptation to environmental conditions. Aurora-A, which is regulated by the system of IDRs we will study in this sLoLa, represents an exciting exemplar protein as it plays a central role in the cell cycle and therefore cancer. The scale of the opportunity here alone is significant: every year, over 250,000 people in England are diagnosed with cancer, and 130,000 die from the disease. Annual NHS costs for cancer services are £5 billion, but the cost to society as a whole - including costs for loss of productivity is £18.3 billion.
To date, the study of IDRs has been largely restricted to in vitro studies, focussed largely on pairwise interactions and isolated sub-topics e.g. role in signalling or disorder-order transitions. We will develop a comprehensive approach for Aurora-A IDRs, but also applicable to the broader IDR challenge. This will be made available for adoption by end-users to explore and validate novel targets, in Aurora-A biology and beyond, and provide starting points for therapeutics, diagnostics and biomarkers development. Insights derived from this sLoLa could ultimately have significant long term societal and economic impact through drug-discovery leading to improved health outcomes and ability to engineer plants/crops for food production, whilst participation will contribute to people and talent development needed to address this challenge more broadly.
Immediate impact will be:
(a) Highly skilled interdisciplinary researchers, who will develop high level knowledge of IDRs framed through scientific skills in structural biology, chemical biology, genetics and cell biology. The highly interdisciplinary nature of this project will provide a unique training and development opportunity for them, as they will need to develop close understanding of the different disciplines in order to advance the project. They will develop a range of transferable skills, and be equipped to work in academic or industrial research settings.
(b) To foster new collaborations between investigators within the team, support career development (particularly early career investigators) and embed team-based working in the mind-set of participants to support the pursuit of grand-challenges research.
(c) The general public, from secondary school-aged children upwards, who will benefit through events designed to provide an increased scientific understanding of well-known diseases and protein dynamics and interactions. For instance schoolchildren through School visits, Leeds Festival of Science and the Astbury Conversation and the University of Leeds Be Curious research showcase, helping to stimulate interest in science as a career choice.
Medium to longer term impact will be:
(d) The international academic and end user community, who will benefit from (i) the integrated fundamental knowledge generated on IDR structural dynamics, molecular mechanism and cellular function (ii) the generic framework we exemplify for pursuing analyses and manipulation of IDRs (iii) the enhanced understanding of Aurora-A biology.
(e) New opportunities for the pharmaceutical and life sciences industries, who will benefit from new understanding and novel starting points for development of drugs and diagnostic approaches. Aurora-A is a compelling anticancer drug target but there has been a failure to deliver a clinical drug due to the side-effects of disrupting all Aurora-A activity. Healthcare services could benefit through novel treatment options for cancer, thus benefitting clinicians and patients through improved treatment options and outcomes. Society and the economy ultimately benefit from improved survival rates, return to economic activity/social integration for patients and reduced burden on healthcare service