Award details

Mapping the interaction of Hsp90 with a new class of client protein: Sgt1-dependent recruitment of a kinetochore protein to Hsp90

Reference	BB/J007595/1
Principal Investigator / Supervisor	Dr Cara Vaughan
Co-Investigators / Co-Supervisors
Institution	Birkbeck College
Department	Biological Sciences
Funding type	Research
Value (£)	405,646
Status	Completed
Type	Research Grant
Start date	15/10/2012
End date	06/11/2016
Duration	49 months

Abstract

The molecular chaperone Hsp90 is an essential protein involved in many signaling pathways and is a drug target for several diseases including cancer. Many aspects of its function are poorly described at a molecular level, including how cochaperones link Hsp90 to substrate proteins and how different functional classes of substrate interact with Hsp90. The development of novel therapeutics will rely on gaining this kind of detailed mechanistic understanding of Hsp90 function. The cochaperone Sgt1 recruits several proteins involved in kinetochore assembly and function to Hsp90 for activation. The kinetochore is the complex of proteins that bridges sister chromatids and microtubules in mitosis; it is essential for fidelity of genetic propagation during cell division. This project aims to contribute to a molecular description of the mechanisms involved in kinetochore assembly by focusing on the protein Ctf13, a protein of the yeast kinetochore that requires Hsp90 and Sgt1 function for its activation and the subsequent assembly of a functional kinetochore. The interaction between Ctf13 and Hsp90 is bridged by the cochaperone Sgt1 and the kinetochore protein Skp1, which forms a heterodimer with Ctf13. The goal of this project is to determine the structure of the complex formed by these proteins and from this structure, to understand how Sgt1 and Skp1 facilitate the activation of Ctf13 by Hsp90. We will use protein crystallography to gain an atomic resolution structure of the Sgt1-Skp1 interaction, and single particle electron microscopy to determine the structure of the complex formed by these proteins with Hsp90 and Ctf13. This structural work will be complemented by biophysical studies, to probe the role of phosphorylation of Sgt1 in influencing oligomerisation of Sgt1 and its interaction with Skp1.

Summary

The function of a protein is determined to a large extent by its structure. The correct structure or 'fold' of a protein is therefore essential for that protein to carry out the specific activity for which it evolved. Misfolded proteins often become inactive or are hyperactive, and are frequently the cause of disease. For many proteins the ability to form the correct fold is something that is innate, that is, it is encoded within the amino acids that make up the protein itself. However some proteins cannot spontaneously fold and require the help of other proteins known as chaperones. These chaperones accompany or guide the protein to the correct fold so the protein can function properly. Chaperones are therefore essential for all living cells. We aim to understand how one such molecular chaperone, called Hsp90, helps proteins to achieve the correct fold. We are particularly interested in this chaperone since many proteins that require Hsp90 for their final structure are involved in diseases, including cancer. The results from this work can therefore be used to develop new drugs that prevent these diseases. We have focused on one protein, called Ctf13 that is involved in cell division. Cell division is the process by which cells replicate, for example to replace cells that are lost through wear and tear, to generate new cells with different functions (for example during the growth and development of a foetus) or during the growth of a tumour in cancer. Two other proteins are required in addition to Hsp90, to allow Ctf13 to fold properly: a helper-protein called Sgt1, which Hsp90 needs to recognize Ctf13, and a bridging protein called Skp1, that bridges the interaction between the helper protein and Ctf13. In order for Ctf13 to attain the correct structure all four proteins must interact simultaneously with each other in a multi-protein complex. The aim of this project is to determine the structure of the complex formed by these proteins. We will do this in several steps. Initially we will determine the atomic structure of the complex formed by the helper-protein and the bridging-protein alone. We will then add the remaining components to form the complete complex, and determine the structure of this using a technique that allows direct visualization of the proteins in a microscope using a beam of electrons instead of a beam of light. Analysis of these structures will allow us to understand more about how Hsp90 interacts with these proteins, and this information can be used to develop new drugs that inhibit Hsp90 function, preventing Hsp90 from 'chaperoning' proteins that are involved in disease.

Impact Summary

The two main beneficiaries of this research beyond academic users are the pharmaceutical industry and the wider public. More than 10 Hsp90 inhibitors are currently in clinical trials and initial results are promising in a range of cancers, including prostate, breast, melanoma and multiple myeloma, confirming Hsp90 as a valid cancer therapeutic target. These drugs inhibit chaperone function by directly competing with ATP-binding, inhibiting the ATPase cycle that is required for client protein activation. Direct inhibition of Hsp90's interaction with client proteins is another suitable method of abrogating Hsp90 function, however development of suitable inhibitors requires details of these interactions at a molecular level. The results from this research will contribute to this poorly understood area of Hsp90 structural biology. Pharmaceutical companies with anti-cancer drug development programs that target inhibition of Hsp90 will therefore be users of this research. This includes Astex Therapeutics and Vernalis in the UK and Novartis, Synta Pharmaceuticals, MedImmune, Infinity Pharmaceuticals and NexGenix in the US. In the long term, by potentially contributing to the development of new anti-cancer therapeutics, this research will contribute to the health of the wider public. At present 1 in 3 people in the UK will develop cancer during their lifetime and 1 in 4 deaths in the UK are currently caused by cancer. A larger battery of anti-cancer drugs will therefore contribute to the long term to the health of the nation.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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