The molecular machine controlling cell growth
Professor David Barford leads a team within the Section of Structural Biology that has recently published work in the prestigious scientific journal Nature* examining the structure of a protein complex known as the anaphase-promoting complex (APC/C).
APC/C is a very large molecule created by joining together 13 different proteins into a kind of molecular machine involved in regulating the cycle cell – a series of complex events that lead to the duplication (replication) of a cell. APC/C controls cell division by recognising and tagging proteins by attaching a long chain of a protein called ubiquitin This polyubiquitin chain triggers degradation of the protein by a process called ubiquitin-mediated proteolysis.
In cancer, the cell cycle is disrupted, allowing cells to multiply out of control. APC/C is known to be involved in the development of some cancers such as breast and bowel. In order for scientists to design a drug that could effectively target APC/C, they need to know its precise shape, how the subunit proteins fitted together and exactly how it tags proteins for degradation.
The first of two papers published in Nature, showed that most of the 13 subunits of APC/C form a scaffold or framework defining the complex’s triangular shape, but others form the key ‘sockets’ for the docking of substrates. To read more about this work see 'Looking Inside The Cell: 3-D modelling a Key Molecule Involved in Cancer Development'.
In the second Nature paper, the team set out to define where and how all the essential units of the protein fitted together – a bit like putting together a 3D jigsaw puzzle – building the first detailed structural model of the APC/C.
Synthesising APC/C
First, the scientists needed to develop an ‘artificial’ system to produce APC/C that would allow them to remove some of the essential APC/C subunits which cannot be deleted in the normal cell. They achieved this by genetically engineering viruses containing DNA that coded for all APC/C subunits, and then infecting insect cells with these viruses. As the insect cells grow they produce complete APC/C molecules. The system produced nearly 200 times as much APC/C as a normal cell, and the team were able to show that the synthesised molecule looked and behaved in a similar way to the natural APC/C.
Defining the organisation and structure of APC/C: Subtracting parts to picture the whole
Firstly, the team wanted to determine the size of the complex and the copy number of each subunit within the APC/C. In collaboration with Professor Carol Robinson’s group at the University of Oxford, the researchers used mass spectrometry (an extremely accurate way to weigh molecules) to determine the absolute mass of the complex for the first time.
Next the team set about working out how the 13 proteins fitted together. Using the system they developed to synthesise APC/C, the researchers were able to generate a series of APC/C sub-complexes, each one with one or more of the 13 subunits missing. Using an electron microscope, they took a picture of each sub-complex and superimposed and subtracted it from the complete molecule. In this way they were able to piece together where each subunit was located within the framework of the whole complex, piece by piece, reconstructing the entire complex. The reconstruction of the complex allowed the scientists to determine which of the 13 proteins were important for the action of the molecule, such as recognising and targeting proteins for degradation, and which of the subunits formed the scaffold of the complex.
Lead author Anne Schreiber, a PhD student in Professor Barford’s team, says: ”The methods we have used here could be used to build a molecular model for other large complexes, and help us learn and understand more about the structure and function of other important molecular machines within the cell.”
New structure, new understanding
The new 3D model provides detail of APC/C down to the molecular level and gives scientists insights into how this molecular machine regulates cell division.
Professor Barford says: “The new model allows us to understand how the molecule interacts with other enzymes and proteins to control cell division. This work is a major step towards beginning to develop therapies that control APC/C activity in cancer.”
*’Structural Basis for the subunit assembly of the anaphase promoting complex’ was published online in Nature on 10 February 2011.