Proteins form the labor force of our cells, carrying out the majority of cellular functions. The Sixma lab has a particular interest in proteins involved in ubiquitin conjugation. Sixma: “The addition and removal and removal of the small ubiquitin peptide, or ubiquitination, is an important signaling system. This type of communication is critical for almost any pathway in the eukaryotic cell. Because it is so important, the enzymes involved in ubiquitin signaling can provide a druggable vulnerability in cancer cells. We study several enzymes that add or remove ubiquitin to other proteins. An example is the work of Niels Keijzer. He has been a PhD student in my group for almost three years now, investigating ubiquitination and deubiquitination of the PCNA protein. PCNA is involved in DNA replication.”
Unrepaired damage
Replication of DNA is one of the key steps in each cell division. During the process of replication, it is important that the DNA is copied with as few errors as possible. Those errors can occur because of damage or lesions to the DNA. Our cells acquire tens of thousands of such DNA lesions per day. This may be caused by ultraviolet light, toxic chemicals, or tobacco products, but also by internal factors such as the reactive oxygen compounds that arise as by-products in normal physiological processes. To prevent our cells from copying these lesions during DNA replication, our cells have mechanisms to detect DNA lesions, signal their presence and ensure their repair. Cells that are defective in these DNA repair mechanisms are highly sensitive to DNA damaging agents and unrepaired damage may result in the development of cancer.
Key step in cell division
Keijzer: “When the DNA replication machinery encounters DNA damage, it will stall. The protein I study, PCNA, will then become ubiquitinated and consequently will adopt a new role: helping the replication machinery bypass the damage. Other proteins will repair the damage later. At a certain point, the ubiquitin needs to be removed from PCNA again to switch it back to its normal function. A key factor in removing ubiquitin and switching PCNA back to normal replication is the protein ‘USP1’. I am eager to find out how USP1 is doing this and how that is regulated and for that we use a plethora of state-of-the-art structural biology technologies.”
Tricky job
The Sixma lab uses structural biology including cryogenic electron microscopy (cryo-EM), X-ray crystallography, nuclear magnetic resonance spectroscopy, and – since the appearance of the Alphafold Database this summer – artificial intelligence. Single particle analysis by cryo-EM allows researchers to determine biomolecular structures at near-atomic resolution. “These microscopes allow us to zoom into the atomic details of protein molecules. I am using simplified in vitro systems in the cryo-EM-studies. Basically, you freeze a tiny drop of liquid that contains a high concentration of purified proteins, in my case USP1 and PCNA. We are constantly trying to improve our protocols because they contain many laborious steps”, says Keijzer. Sixma agrees: “Preparing the samples and freezing them correctly is a tricky job. For instance, if you drop the tweezers once, you have to buy a new pair. And it takes time to learn how to handle the fragile little grids that hold the sample in the microscope.”
