Interview

Comprehending Protein Complexes

Interview with molecular biologist Lori Passmore

19.3.2014 | Dr. Lori Passmore studies complex multi-protein structures in a Cambridge lab with a history of producing winners of the Nobel Prize.
AcademiaNet: You've spent your research career in molecular biology studying complex multi-protein structures that are involved in regulating cell division and protein production. What makes you interested in these large protein complexes versus single proteins?

Dr. Lori Passmore: Usually proteins work in concert with lots of other proteins. Although studying them in isolation gives us important information about their enzymatic mechanisms, we can only understand their regulation and how they work in a physiological context when we study them as intact complexes.

But how do you figure out what a particular complex does: do you try to attribute functions to the different proteins in the complex or do you look at the complex as a whole?

We do a little bit of both, generally using yeast as a model organism. We make genetic mutations or delete protein subunits within the yeast and then either study what happens in vivo in the yeast cells or purify the protein complexes to understand their structure and biochemical properties in vitro.

Yeast is a good model organism for any eukaryote. Eukyaryotes are all organisms whose cells contain a nucleus, including humans. Lots of the really fundamental processes are similar, so we can draw parallels between what we discover in yeast and what happens in humans. And we've also started to study higher eukaryotic systems to try to validate what we've seen in yeast.
Dr. Lori Passmore
Bild vergrößern
(© MRC Cambridge)


Dr. Lori Passmore | at the MRC Laboratory of Molecular Biology in Cambridge

Speaking of humans, one area of your research are the multi-protein complexes involved in Fanconi anaemia, a genetic disease that predisposes people to cancers, such as leukaemia, and bone marrow failure. What role do complex proteins have in this disease?

The Fanconi anaemia core complex is a large ubiquitin ligase, an enzyme involved in the DNA damage response. Ubiquitination happens in response to DNA damage, and that recruits DNA repair proteins. When that process is defective, people get Fanconi anaemia, resulting in bone marrow failure and developmental abnormalities. We're trying to understand how the core complex performs its functions and what goes wrong in disease by purifying the complex and studying its biochemical and structural properties.

Another key area of your research at the MRC Laboratory of Molecular Biology in Cambridge are the protein complexes that add and remove poly(A) tails from the 3' ends of mRNA. What is the importance of poly(A) tails in cell function?

Poly(A) tails are present on almost every protein-coding mRNA. Messenger RNA, or mRNA, convey genetic information from DNA in the nucleus to the ribosome, where they specify the amino acid sequence of proteins. Poly(A) tails are important for the stability of the mRNA and regulate their translation. In short, they act to fine tune gene expression. For example, a shortened poly(A) tail can repress translation of the mRNA or cause mRNA to decay.

What are the large protein complexes that are involved in this process and how do they work?

One of the protein complexes is called "cleavage and polyadenylation factor", or CPF, which has 15 different protein subunits. It functions co-transcriptionally to process the 3' end of the mRNA and add a poly(A) tail. That process triggers maturation of the mRNA, allowing it to be exported from the nucleus of the cell to the cytoplasm, and also triggers termination of transcription. This happens with almost every mRNA in the cell.

We're also studying the complexes that remove poly(A) tails. The Ccr4-Not complex is composed of up to nine different proteins. It can be specifically recruited to mRNAs, where it degrades the poly(A) tail but not the rest of the mRNA. We don't understand why so many protein subunits are found in these complexes, which perform relatively simple biochemical reactions. We suspect that their complexity is related to the intricate regulation of these fundamental and important processes.

What techniques and equipment do you use in your labs to make sense of these complex proteins?

Cryo-EM graph of Eukaryotic Ribosomes
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Cryo-EM graph of Eukaryotic Ribosomes
Generally we work out methods to purify the intact complexes and once we have done that, we can study them using biochemical techniques to understand how they assemble, which proteins associate with which other proteins, and how they're regulated. But we also study the structure of proteins by using electron microscopy. To do this, we rapidly freeze the protein complexes to liquid nitrogen temperatures and we image them using an electron microscope, this process is called cryo-EM. With this technique you can get to near atomic resolution, and you're able to understand the structure of these large multi-protein complexes. We also use x-ray crystallography and nuclear magnetic resonance imaging to get a complete picture.

Both the hardware and software for biological electron microscopy are still developing, and there has been a lot of progress over the past few years. My lab is contributing to this by developing new methods for sample preparation and imaging. It's really exciting to be in a field that is changing so rapidly, where we can do experiments that were not possible only a few years ago.

You completed your undergraduate studies in Canada at the University of British Columbia, but have spent the rest of your career in the UK. What first brought you to England and has since kept you here?

Originally I came to England for the science. I was interested in the projects that David Barford was initiating in his lab at the Institute of Cancer Research in London, in particular on the anaphase promoting complex/cyclosome, or APC/C. It was really exciting and a great chance to learn new techniques and experience a new environment. Once I had arrived here, I began to really appreciate the funding for basic, fundamental science in the UK and in Europe, and the support available for young investigators starting out. That has been important for me staying here.

The lab you work in has produced 13 Nobel Prizewinning scientists, including Francis Crick and James Watson, who won the prize in 1962 for determining the structure of DNA. Is this history perhaps a little intimidating?

I think it is really inspiring. My post-doctoral advisor Venki Ramakrishnan was awarded the Nobel Prize in Chemistry in 2009, which was a really exciting time. Everyone here is pretty down to earth, approachable and loves sinking their teeth into an important scientific problem. The Laboratory of Molecular Biology is a fantastic place to be, it's very collaborative, and it supports basic fundamental research. And we have fantastic facilities, so it has been instrumental in helping me get started as an independent scientist. Like most people here I see the history of the lab as inspiring rather than intimidating.

Dear Dr. Passmore, thank you very much for this interesting interview!

Interview: Helen Jaques   (© AcademiaNet)

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