Tandem repeat proteins
A major aim of our current research is to define, using a battery of techniques spanning single molecule methods, biophysics, chemical biology and analysis in cellulo and in silico, the conformational transitions that proteins undergo during the different stages of their life cycle - biosynthesis, folding, localisation, assembly and degradation - and how they are guided by the cellular machinery. In order to achieve our goals a major focus of our efforts is the class of proteins known as tandem repeat proteins, which constitute almost 20% of proteins encoded in the human genome. They comprise small structural units repeated multiple times in tandem to form non-globular, elongated structures that provide extended surfaces for molecular recognition (Fig. 1). Our group and others have shown that the simple modular, one-dimensional architecture of tandem repeat proteins gives them distinctive properties compared with globular proteins and makes it uniquely straightforward to map the energetics of their structures and to rationally redesign their stability, folding and molecular recognition. This class of proteins is thus an exceptionally sensitive and versatile tool that we are now in a position to exploit to dissect otherwise intractable cellular mechanisms (Fig. 2).
We are focusing on ankyrin, HEAT and ARM repeat proteins that play important roles in disease, in particular cancer, and we are using the insights we obtain to develop therapeutic strategies for targeting them (e.g. Fig. 3). Ankyrin repeats have also been identified as targets in a range of diseases in addition to cancer. For example, antagonists of ankyrin repeat ion channels have shown potential in pain relief and in treating respiratory diseases, chronic acid reflux and esophageal hypersensitivity.
The properties of the individual units of tandem repeat proteins can be tailored by design and they can then be mixed and matched in a modular fashion to create artificial proteins with predictable properties (stability, binding, etc) and also with multi-functionality; such a degree of rational engineering is not possible with globular proteins. We are interested in exploiting this extraordinary design-ability in medicine and biotechnology.
Molecular pathology of cancer-associated missense mutations?
Our previous in vitro analysis suggests that many missense mutations in cancer-causing tumour suppressors p16 (an ankyrin repeat protein) and BRCA1 drastically destabilise the structures (Fig. 4), thereby causing loss of function. Our aim is now to understand how proteins respond to mutation when they are surrounded by the complex environment of the cell.
Rowling, P.J.E., Cook, R., Itzhaki, L.S. Towards classification of BRCA1 missense variants using a biophysical approach. J. Biol. Chem. 285: 20080-7 (2010).
Serquera, D., Lee, W., Settani, G., Paci, E., Marszalek, P., Itzhaki, L.S. Mechanical unfolding of an ankyrin repeat protein. Biophys. J. 98: 1294 301 (2010).
Werbeck, N.D., Rowling, P.J., Chellamuthu, V.R., Itzhaki, L.S. Shifting transition states in the unfolding of a large ankyrin repeat protein. Proc. Natl. Acad. Sci. USA 105: 9982-7 (2008).
Lowe, A.R., Itzhaki, L.S. Rational redesign of the folding pathway of a modular protein. Proc. Natl. Acad. Sci. USA 104: 2679-84 (2007).