Cargo recognition by kinesin-1 and its role in activation of transport

Cells possess many specialised components that must be in the right place at the right time to fulfil their function. After their use, these components must be transported away for recycling or degradation. Mis-regulation or disruption of these transport processes can contribute to many human diseases ranging from neurodegenerative conditions such as Alzheimer's disease to cancer and even contribute to viral infections by HIV-1 or bacterial infections such as Salmonella. To move components around, cells use a transport system composed of a network of cables known as the microtubule network. Much like a railway network, these cables link together regions of the cell. Cells possess vehicles that travel along this network known as molecular motors, of which our proposed motor of study, kinesin-1, is one of the most important. These motors can selectively attach to cellular components and move them on the microtubule network. Despite their importance across so many areas of cell biology, we lack a proper understanding of how these motors recognise the cargo that they carry.

The Dodding and Steiner groups at the Randall Division of Cell and Molecular Biophysics at King's College have collaborated to tackle this important problem and recently managed to take a big step forward. We have shown how kinesin-1 recognizes one of its cargoes known as SKIP - a cellular protein usurped by Salmonella for its kinesin-1 binding function. This breakthrough now gives us the exciting opportunity to study how kinesin-1 recognises is many other cargoes with diverse functions. We propose to do that here.

We also know that cargoes themselves can control when motors attach to the transport network and move. In the absence of cargo, motors are inactive and don't move however when cargo are attached a switch occurs which allows motors to attach to the transport network and move. This is perhaps analogous to giving a taxi driver a signal to drive off when you are safely in the car. Despite many years of study, how this switch works is not understood at a molecular level. Our new data suggest an unanticipated mechanism by which this might occur and we propose to explore these exciting new ideas here.

Our approach will combine cellular imaging, X-ray structural analysis and biochemical/biophysical techniques to obtain a full range of biological insights. In the long term we consider it possible that an ability to modulate motor-cargo interactions and motor activity may be gained from these studies could be useful for treatment of a range of human diseases.

Kinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Despite this we still know relatively little about how kinesins specifically recognise the cargoes that they carry from the milieu of proteins in the cytoplasm nor how binding of that cargo can control the activity of the motor. Recent studies have uncovered that the light chain TPR domain of the ubiquitous motor kinesin-1 (KLCTPR) can recognise short peptide stretches within relatively disordered regions of its targets. These peptides are characterised by a tryptophan residue flanked by acidic residues (e.g. EWD) and are found in many kinesin-1 cargoes. We call these motifs 'W-acidic'. A collaborative effort between the Dodding and Steiner groups has resulted a major step forward in understanding recognition mechanisms of W-acidic motifs. We have recently published the crystal structure of a kinesin-cargo complex where we show how KLC2TPR recognises a W-acidic motif from the SKIP cargo protein. We propose to build on that success to further our understanding of recognition mechanisms. We will investigate how interaction with W-acidic cargo motifs couples to controlling the activity of the kinesin-1 motor. We will also study how KLC specifically recognises motifs in cargoes that don't carry the W-acidic consensus such as JIP1, with the overall aim of fully explaining the molecular basis for cargo recognition. Finally, we will begin to explore how ternary interactions between kinesin-1 and cargo bearing W-acidic motifs contribute to binding affinity and how that impacts on transport. Combined, these three strands will lay the ground for a complete picture of the process kinesin-1 cargo recognition, activation and transport. Our approach will combine X-ray structure and biophysical techniques and with analysis of human cells in culture to obtain a full range of biological insights.

Due to the central importance of microtubule motor proteins for all forms of eukaryotic life, a core understanding of their basic function will impact across a wide breadth of biomedical science. Moreover, the study of this topic may in the long term, contribute to the health and wellbeing of the population.

i. Pharmaceutical and biotechnology industries

Work described in the proposal examines the molecular interface between kinesin-1 and cellular, viral and bacterial proteins associated with human diseases. These include Salmonella and poxvirus infections as well as neurodegenerative and hereditary conditions. As well as providing basic knowledge that is essential for an understanding of these diseases, an important aim of our research in the long term and for which this proposal will lay an important foundation is to determine whether disruption of this interface could selectively block transport of cellular cargoes associated with human diseases. This may lead to the commercialisation of the scientific knowledge obtained from this proposal and/or the formation of spin out companies, thereby contributing toward wealth creation and economic prosperity of the nation. This would also serve to attract R&D investment from global business.

ii. Patients who suffer from diseases where microtubule transport is impaired or usurped and clinicians who treat those diseases
As indicated above, we our work address a crucial questions that lies at the heart of cell biology and disease. Whether this involves the hijacking of the transport system in the case of pathogen infection or whether mis-regulation of transport is an important links to the disease as in the case of Alzheimers, our research of this molecular interface will add to the knowledge of understanding of these diseases and offers the long term hope of targeting the motor/cargo interface which will benefit many. This maps onto the BBSRC aim of providing understanding of "Basic Bioscience Underpinning Health" and because of the role of kinesin based microtubule transport of several of the cargos on which we will work in neurodegenerative disorders such as Alzheimer's disease, fits within the "Ageing research: lifelong health and wellbeing" strategic priority area.

iii. The wider public

The wider public will gain from an increased understanding of how the human body works on a molecular level. The engagement of the public with academic science has become a priority and we will take steps as outlined in pathways to impact to ensure that our work is communicated to the widest non-academic audience possible.

iv Staff funded by this project

Staff working on this project will receive a thorough training in molecular, cellular, and structural biology in one of the UK's leading scientific institutions. It is highly likely that they will use this knowledge to make further contributions either in academic or industry, which will in turn benefit the UK economy.

Details of how we propose to maximize these impacts for these beneficiaries are described in our 'pathways to impact statement'.