Dougan - Protein homeostasis in health and disease
Protein homeostasis (commonly known as proteostasis) is a fundamental biological process of all cells. It maintains protein quality within the cell and plays a crucial role in the overall health of an organism. At the front-line of this battle to maintain correct protein function within the cell, are proteins known as molecular chaperones and proteases. These "defence" proteins, not only play an important during the normal day-to-day running of a cell, but they also play a crucial role during periods of stress.
As such molecular chaperones and proteases are often thought to underpin the cells ability to survive hostile conditions and understanding how these proteins work to protect the cell may have important implications not only in disarming pathogens (to make them more sensitive to stressful conditions inside the host) but also in developing strategies to assist the cell in overcoming metabolic or age-related diseases.
Regulation of AAA+ proteolytic machines
AAA+ (ATPases associated with a variety of cellular activities) proteases are a ubiquitous group of proteins that are found in all kingdoms of life, from bacteria to mammals. They form large molecular machines that use the energy provided from hydrolysis of ATP, to unfold target proteins and feed them into a degradation chamber. Generally, they consist of an AAA+ unfoldase (ATPase) component (e.g. ClpX) in association with a peptidase component (e.g. ClpP).
The key steps in the protein degradation pathway are (1) substrate recognition and delivery, (2) unfolding and translocation, and (3) proteolysis. The unfoldase component is largely responsible for substrate recognition, however the specificity of these machines may often be extended (or altered) by specific AAA+ co-factors known as adaptor proteins.
A major focus of our research involves the molecular dissection of these machines, how they work, the substrates they recognise and the proteins that influence their substrate-binding repertoire. Currently we are studying a number of different AAA+ proteins, from a variety of bacterial species (both pathogenic and non-pathogenic) and, in collaboration with Dr Kaye Truscott, from mammalian mitochondria.
The N-end rule pathway
The N-end rule pathway is a ubiquitous protein degradation pathway found in bacteria, yeast and mammals, which relates the half-life of a protein to the identity of its N-terminal residue. Some residues (red) are considered 'stabilizing' when located at the N-terminus of a protein (i.e. the protein is not degraded), while other N-terminal residues (yellow or green) are 'destabilizing' (i.e. trigger rapid turnover of the protein).
Previously, we have shown that the adaptor protein ClpS is essential component of the pathway, required for the recognition (and ClpAP-mediated degradation) of proteins bearing an N-terminal destabilising residue in E. coli (Erbse et al., 2006). We have also identified several natural substrates of this pathway in E. coli (Ninnis et al., 2009) and in collaboration with colleagues in Germany, we have defined to atomic detail, the nature of this elegant interaction between the ClpS and the N-degron (Schuenemann et al., 2009).
Currently we are studying several novel components of the N-end rule pathway, not only in E. coli but also in pathogenic bacteria and mammals.
Stress response pathways
Stress response pathways, such as the general stress response pathway, play important roles in bacterial virulence. In the model Gram-negative bacterium, Escherichia coli the general stress response pathway is regulated by the transcription factor, SigmaS. The activity of which, is controlled not only at the transcriptional and translational level but also at the level of protein stability. Under normal cellular conditions, SigmaS is rapidly degraded by the protease ClpXP, but only in the presence of the adaptor protein RssB. Interestingly, the activity of RssB is regulated not only by phosphorylation, but also by several "stress inducible" proteins (e.g. IraD, which responds to DNA damage) known as anti-adaptors.
Currently, we are dissecting the molecular details of the interaction between SigmaS and RssB, and its delivery to ClpXP. We are also interested in the mechanism of inhibition of the various different anti-adaptor proteins. Developing a detailed understanding of these interactions could provide the opportunity to develop novel antibiotics against pathogenic bacteria.
Our group is also interested in the role of several related mitochondrial proteases (LONM, ClpXP and m-AAA) and how these proteins contribute to stress response pathways in humans, such as the mitochondrial unfolded protein response. These projects are performed in collaboration with Dr Kaye Truscott.